ARTICLE
pubs.acs.org/IC
Geometric and Electrostatic Study of the [4Fe-4S] Cluster of
Adenosine-50-Phosphosulfate Reductase from Broken Symmetry
Density Functional Calculations and Extended X-ray Absorption
Fine Structure Spectroscopy
Devayani P. Bhave,† Wen-Ge Han,|| Samuel Pazicni,‡ James E. Penner-Hahn,‡,§ Kate S. Carroll,*,†,^ and
Louis Noodleman*,||
Chemical Biology Graduate Program, ‡Department of Chemistry, and §Department of Biophysics, University of Michigan, Ann Arbor,
Michigan 48109-2216, United States
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States
^
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States
)
†
bS Supporting Information
ABSTRACT:
Adenosine-50 -phosphosulfate reductase (APSR) is an ironsulfur protein that catalyzes the reduction of adenosine-50 -phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X∼80-CXXC motif, and the cluster is essential
for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the ironsulfur cluster in APS
reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and
extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and
electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe4S]2þ state in both native and substrate-bound APSR while EXAFS data recorded at ∼0.1 Å resolution indicates that there is no
significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other
hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APSbound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests
a role for the unique coordination in facilitating a compact geometric structure and “fine-tuning” the electronic structure to prevent
reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that
Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.
1. INTRODUCTION
In plants and many species of bacteria such as Mycobacterium
tuberculosis (Mt) and Pseudomonas aeruginosa (Pa), de novo
synthesis of cysteine occurs via the sulfate assimilation pathway.1
In this pathway, inorganic sulfate is activated to form adenosine-50 phosphosulfate (APS), which is subsequently reduced to sulfite
and then sulfide, and incorporated into cysteine.2,3 The first
committed step in sulfate assimilation is carried out by the enzyme,
adenosine-50 -phosphosulfate reductase (APSR), which catalyzes
the reduction of APS to sulfite and adenosine-50 -monophosphate
r 2011 American Chemical Society
(AMP) (Scheme 1) using reducing equivalents from thioredoxin
(Trx), a protein cofactor.1,46 APSR has been shown to be
essential for survival of bacteria in the latent phase of tuberculosis
infection,7 and since there is no human homologue of APSR, it
represents a promising drug target for antibacterial therapy.8
APSRs from M. tuberculosis (Mt-APSR) and P. aeruginosa (PaAPSR) are related by high sequence homology (27.2% of
Received: March 4, 2011
Published: June 16, 2011
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Scheme 1. Reaction Catalyzed by APSR
Figure 1. Proposed mechanism of APSR. APSR reduces adenosine
50 -phosphosulfate (APS) to sulfite and adenosine 50 -monosulfate (AMP)
using reducing equivalents from the protein cofactor, thioredoxin (Trx).
sequence identity and 41.4% of sequence similarity, Supporting
Information, Figure 1), particularly in the residues that line the
active site.9 The mechanism of APSR involves a nucleophilic
attack by the catalytic Cys256 (residue numbers throughout
manuscript correspond to the Pa-APSR sequence) in the
C-terminal tail of APSR on APS to form an enzyme S-sulfocysteine intermediate, E-Cys-Sγ-SO3, which is then reduced to
sulfite and AMP through intermolecular thioldisulfide exchange with Trx (Figure 1).4 It is likely that in the initial
Michaelis complex APS binds to other residues, changing their
resulting mobility within the substrate-binding pocket; this could
have important mechanistic implications.
From a structural perspective, APSR is an ironsulfur protein
with a conserved CC-X∼80-CXXC motif, correlated with the
presence of a [4Fe-4S] cluster. The cluster has been shown to
be essential for catalytic activity in both plant and bacterial
APSRs,3,1012 but the exact role of the cluster in APS reduction
remains unknown. Interestingly, studies by Carroll et al. have
shown that the [4Fe-4S]2þ cluster in APSR does not undergo
redox changes during the catalytic cycle.4 The 2.7 Å crystal
structure of Pa-APSR bound to substrate13 shows the ironsulfur
cluster coordinated by Cys228 and Cys231, positioned at the tip of
a β-loop, and a special tandem pair, Cys139 and Cys140 within a
kinked helix, R6 (Figure 2A). Helix R6 is kinked where Lys144 is
oriented into the active site. Among interactions of the cluster,
there are four charged and/or polar NH 3 3 3 S or OH 3 3 3 S hydrogen bonds involving side chains of absolutely conserved residues
(Figure 2B and C). The CysCys motif interacts with a pair of basic
residues, Arg143 and Lys144. In addition, Cys140 hydrogen bonds
to His136. Other interactions with the ironsulfur cluster involve
the side chains of Thr87 and Trp246. The phosphosulfate group of
APS is positioned at a distance of approximately 7 Å to the iron
site, which coordinates to Sγ-Cys140, and as such, the sulfate
moiety is not in direct contact with the [4Fe-4S] cluster. Interestingly, however, both cluster and substrate interact with Lys144
(Figure 2C).
Coordination by sequential cysteines is highly unusual for
[4Fe-4S] clusters and has been characterized in only one other
crystal structure, the NuoB subunit of respiratory complex I.14
The tandem cysteines also reside within an R-helix, and the
subunit of NuoB exhibits substrate-induced conformational
changes.15 In APSR, constraints imposed by the tandem cysteine
coordination do not affect the tetrahedral symmetry of the
cluster, but the side chain of Cys140 is distorted, resulting in
steric clashes between the CR proton and an inorganic sulfur
atom of the cluster.13
In addition to the structural information available, based on
differences in cysteine reactivity and cluster stability, biochemical
and mass spectrometric studies with Mt-APSR have suggested a
structural rearrangement in the S-sulfocysteine complex and
AMP-bound enzyme relative to free enzyme.16 In fact, solution
kinetics and mass spectrometric studies of Mt-APSR performed
with APS (at concentrations exceeding the Kd of APS and AMP),
have shown that the subsequent formation of the stable E-CysSγ-SO3 intermediate with AMP-bound and C-terminal tail
docked in the active site, prevents cluster degradation and loss
of APSR activity.4,9,16 Furthermore, a comparison of Resonance
Raman spectra of Pa-APSR in the native form and in the Ssulfocysteine form (with AMP bound and the C-terminal tail
docked in the active site of the enzyme) shows an enhancement
in FeSγ(Cys) stretching modes centered near 355 and
369 cm1.17 Recently, we demonstrated that APS binding
induced an increase in intensity and resolution of the EPR signal
of reduced Mt-APSR that was not observed among a panel of
substrate analogues, including adenosine 50 -diphosphate. Additionally, through kinetic and electron paramagnetic resonance
(EPR) studies, Lys144 was identified as a key link between APS
and the ironsulfur cluster.18 M€ossbauer analyses of native MtAPSR confirmed the presence of a [4Fe-4S]2þ cluster; however,
no change was observed in the M€ossbauer spectra of Mt-APSR
comparing samples with and without substrate-binding. Spectroscopic data taken together with known structural and functional
information, implicate the ironsulfur cluster in the catalytic
mechanism of APS reduction.
The goal of this study is to determine high-resolution geometric and electronic structures of the [4Fe-4S] cluster in APSR.
Although the crystal structure of Pa-APSR is a significant advance
in the characterization of this enzyme, the resolution of
the structure (2.7 Å) is close to the FeFe distance within a
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Figure 3. XAS analysis of Mt-APSR. (A) Iron K-edge XANES spectrum. (B) k3 weighted EXAFS (inset) and Fourier transform calculated
over a range of k = 2.3517 Å1.
Figure 2. Environment of the [4Fe-4S] cluster in Pa-APSR.18 (A) The
structure of Pa-APSR bound to substrate APS (subunit B or chain-B).
The [4Fe-4S] cluster is ligated by four cysteine residues at positions 139,
140, 228, and 231. PDB code: 2GOY. (B) Three conserved residues
participate in charged or polar NH 3 3 3 S or OH 3 3 3 S hydrogen bonds to
inorganic S or cysteine Sγ atoms; Thr87, Arg143, and Trp246 (yellow
dashes). PDB code: 2GOY, chain A. (C) Conserved basic residues
Lys144, Arg242, and Arg245 in the active site interact with the
phosphate and sulfate groups of APS (yellow dashes). In the presence
of APS, Lys144 makes a NH 3 3 3 S hydrogen bond to the Cys140-Sγ
atom. Residues that also interact with APS, but are not depicted in this
figure are Arg171 and His259; these residues interact with the Rphosphate group. The shortest distance between a sulfate oxygen atom
and a cysteine sulfur atom coordinated to the [4Fe-4S] cluster is 6.0 Å.
PDB code: 2GOY, chain B.
[4Fe-4S] cluster,19 placing a significant limit on the structural
features that can be visualized in electron density maps calculated
using the X-ray data. Given the unique coordination of the [4Fe-4S]
cluster by two consecutive cysteines, it is important to obtain
direct confirmation of the apparent structure. In particular, the
X-ray structure shows that torsion angles of the Cys139 and
Cys140 side chains are significantly strained, which could affect
details of FeFe and FeS-(Sγ) distances in various states of the
catalytic cycle.13 EPR studies on one-electron reduced Mt-APSR
demonstrate midrange electrostatic interactions involving the
cluster, Lys144 and the substrate that implicate the cluster in
catalysis.18 However, changes in the geometric and electronic
structure of the cluster during catalysis have not been determined
by any structure determination technique. Determining these
high-resolution structures will ultimately further our understanding of the role of the ironsulfur cluster in APS reduction.
The work described here combines Fe K-edge X-ray absorption spectroscopy investigations (both extended X-ray absorption fine structure, EXAFS, and X-ray absorption near-edge
structure, XANES) with density functional theory (DFT) calculations to determine the local geometric structure, spin states,
electrostatic potential charges, and 57Fe M€ossbauer properties of
the [4Fe-4S] cluster of APSR. XANES data confirms that the
cluster is in the [4Fe-4S]2þ state in native Mt-APSR (Figure 3A)
and that there is no detectable change in structure when APS
binds. EXAFS data recorded at ∼0.1 Å resolution (k = 17 Å1)
(Figure 3B) indicates that there is no significant change in the
average FeS and FeFe bond lengths of the [4Fe-4S] cluster
between the native and substrate-bound forms of the protein (see
Supporting Information, Figure 2 for comparison spectra). Since
the EXAFS structure reflects the average over all four irons in the
cluster, it is insensitive to changes at individual sites (e.g., a
decrease in one FeFe distance that is compensated by an
increase in a second FeFe distance). To explore these changes,
DFT calculations starting from the experimental X-ray structure
of Pa-APSR13 were used to provide insight into the subtle
changes within the geometric and electronic structure of the
cluster and how they relate to the role of the [4Fe-4S] cluster in
the mechanism of APS reduction.
2. EXPERIMENTAL MATERIALS AND METHODS
Materials. E. coli BL21(DE3) used for expression was obtained from
Novagen (Bad Soden, Germany). APS was purchased from Biolog Life
Sciences Institute, g95% (Bremen, Germany). AMP and reagents for
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Figure 5. Closer look of the [4Fe-4S] center and the atom labels with
the four cysteine side chains.
Figure 4. DFT optimized quantum cluster model of the [4Fe-4S]
center in APSR without APS. Initial geometry was taken from chain-A of
the X-ray crystal structure (PDB code: 2GOY).13
the buffer were obtained from Sigma-Aldrich (St. Louis, MO) and were
of the highest purity available.
Preparation of Mt-APSR Samples for EXAFS Spectroscopy.
Mt-APSR was purified as detailed in ref 18, concentrated using 10,000
MWCO Amicon Ultra-4 centrifugal filter devices (Millipore Corporation, Billerica, MA), and used at a final concentration of 1 mM in buffer
containing 50 mM TrisHCl, 150 mM NaCl (pH 8.5 at 4 C) and 10%
(v/v) glycerol. Protein concentrations were determined using the
extinction coefficient, ε280 = 36,815 M1cm1, obtained by quantitative
amino acid analysis. Analysis of iron content by inductively coupled
plasma (ICP) mass spectrometry for Mt-APSR indicated that each mole
of protein contained 3.5 ( 0.4 mol of iron, as expected for a [4Fe-4S]
cluster. The iron and sulfur content of APSR is consistent with the
incorporation of four irons and four inorganic sulfides per mol of
protein.4,20 The specific activity of the purified enzyme was 5.2 μM
min1 mg protein1 as determined by an assay using 35S-APS described
in ref 9 and consistent with the previously reported value.4 For samples
with ligand, 1 mM enzyme was incubated with 3 mM ligand for 15 min at
room temperature, prior to loading. All samples were loaded in 1 mm
Lucite cells with 37 μm Kapton windows for X-ray absorption studies,
immediately frozen in liquid nitrogen, and maintained under liquid
nitrogen conditions until data were collected.
EXAFS Measurements and Data Analysis. X-ray absorption
spectra were recorded at the Stanford Synchrotron Radiation Laboratory (beamline 9-3) under dedicated conditions as fluorescence excitation spectra, using a solid-state Ge detector array equipped with a Mn
filter and Soller slits focused on the sample. All channels of each scan
were examined for glitches, and the good channels were averaged for
each sample (two independent samples for each protein composition,
with or without ligand) to give the final spectrum. During data
collection, samples were maintained at a temperature of approximately
263 C using a liquid-helium flow cryostat. As a measure of sample
integrity, XANES spectra measured for the first and last scan of each
sample were compared. No changes were observed over the course of
the data collection. Data were measured with an integration time of 1 s
through the edge, and between 1 and 25 s in the postedge region. For
each sample, between 5 and 10 35-min scans were accumulated, and
each sample was measured in duplicate. The useful fluorescence count
rate per channel was ∼104 counts/second, giving a total of ∼6 106
counts/scan at k = 17 Å1. For energy calibration, the absorption
spectrum of an iron metal foil was measured simultaneously by
transmittance, and the energy was calibrated with reference to the
lowest-energy inflection points of the foil standard, which was assumed
to be 7111.3 eV for iron. EXAFSPAK21 was used to extract and analyze
EXAFS data, using ab initio phase and amplitude parameters calculated
using FEFF version 7.02.22,23 with the initial threshold energy E0 defined
as 7130 eV. Nonlinear least-squares fits of the data used 4 variable
parameters (R and σ2 for each shell) with the amplitude correction factor
and ΔE0 set to 0.9 and 11 eV, respectively, based on fits to model Fe/S
clusters. XANES data were normalized to tabulated absorption
coefficients24 using MBACK.25 The area of the 1s f 3d transition in
the XANES region was calculated by fitting the pre-edge region
(71077118 eV) using the sum of a Gaussian and an arctan function;
for comparison with previously published data, the fitted Gausian area
was normalized to the K-edge jump for Fe (3.556 102 cm2/g).
3. QUANTUM CLUSTER MODELS FOR DFT
CALCULATIONS
3.1. Wild-Type Models with and without Substrate. The
initial geometries for DFT calculations of the wild type APSR
active site models including the [4Fe-4S] cluster were taken from
the X-ray crystal structure (2GOY.pdb, 2.7 Å resolution) of PaAPSR.13 Pa-APSR was serendipitously crystallized such that
density for APS was observed in two of the four monomeric
subunits. Thus the differentially occupied subunits could be used
to compare the geometry and electronic structure of the [4Fe-4S]
cluster in the free and substrate-bound forms of the protein. The
cluster without APS was taken from subunit A (or chain-A) of the
crystal structure (Figure 4). A closer look at the [4Fe-4S-4Cys]
center is shown in Figure 5. The quantum cluster of the active site
with APS was taken from chain-B of the crystal structure
(Figure 6). The main or side chains of Cys139, Cys140, Cys228,
Cys231, Thr87, His136, Arg143, Lys144, Arg242, Arg245, and
Trp246 were also included in the quantum models. To facilitate
calculations by minimizing the number of atoms in the model,
some changes were made to the residues, including cleavage of
certain bonds, addition of link-H atoms,26 and partial inclusion of
neighboring residues to have closed valence. Explicitly, for Thr87,
Cys139, His136, and Cys228, CR-NH was replaced with CR-H.
Since the electron density map shows δþ density on His136, the
His136 side chain was protonated in our calculations. For Gly88,
Gly137, Gly141, and Glu229, HN-CR was included into the
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Figure 6. DFT optimized quantum cluster model of the [4Fe-4S] center in APSR with APS. Initial geometry was taken from chain-B of the X-ray crystal
structure (PDB code: 2GOY).13.
quantum cluster and CR was replaced with H. For Ile142 and
Pro230, CR-CdO was included in the cluster and CR was replaced
with H. For Lys144 and Thr232, CR-CdO was replaced with CRH. For Arg242 and Arg245, Cγ-Cβ was replaced with Cγ-H. Finally
for Trp246, HN-CR-CdO was replaced with HCR-H.
The following three H-bonds are found in the crystal structure
between the inorganic S’s and Sγ’s of the [4Fe-4S-4Cys] cluster
and the protein residues in both chain-A and chain-B: S3 3 3 3
HNε1-Trp246, Cys139-Sγ 3 3 3 HNε-Arg143, and Cys228-Sγ 3 3 3
HOγ1-Thr87. In chain-B where APS is bound, the side chain of
Lys144 H-bonds with both Sγ-Cys140 and O from the sulfate
group of APS. Model cluster for chain-A (Figure 4) has eleven
H-bonds and a total of 211 atoms. By contrast, model cluster for
chain-B (Figure 6) has eighteen H-bonds (of which, 7 involve the
substrate) and a total of 250 atoms.
Without APS, the Arg242 side chain H-bonds with the main chain
CdO group of Arg245 (chain-A, Figure 4). In the presence of APS,
Arg242 rotates to form H-bonds with the phosphate and sulfate
groups of APS. Arg245 also H-bonds with APS (chain-B, Figure 6).
3.2. No-Tandem Models. Since coordination by sequential
cysteines in Pa-APSR is highly unusual for [4Fe-4S] clusters, it is
valuable to determine what changes would occur in the geometric and electronic structure of the cluster if we break the
linkage between Cys139 and Cys140. For this purpose, two
“no-tandem” computational models were constructed by breaking
the HN-CO peptide bond between Cys139 and Cys140 in both
chain-A (without APS) and chain-B (with APS). The CdO
group of Cys139 was replaced by a hydrogen (Figure 7), meanwhile a hydrogen was added to the NH group of Cys140 to fill
the open valence. In the absence of the peptide bond, the number
of atoms in each of the model clusters remained constant.
3.3. K144A Models. As previously mentioned, there is biochemical and spectroscopic evidence to show that the stability
and microenvironment of the cluster in APSR changes upon
substrate binding.4,16,27 This indicates that APS does interact
with the [4Fe-4S] cluster, not by direct contact, but via a network
of electrostatic interactions. Lys144, Arg242, and Arg245 all have
H-bonding interactions with APS, of which Lys144 is positioned
between the [4Fe-4S] cluster and APS. Therefore we constructed the K144A models (for both chain-A and chain-B) by
replacing Lys144 with Ala to investigate changes in the properties
of the [4Fe-4S] cluster without Lys144. Explicitly, the Cβ-Cγ
bond of Lys144 was cut and a proton was added to Cβ to close
valence, resulting in a total of 199 and 238 atoms in the chain-A
and chain-B models respectively.
4. COMPUTATIONAL METHODOLOGY
All density functional spin-unrestricted calculations were performed
using the Amsterdam Density Functional (ADF) package2830 with the
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Figure 7. HN-CO peptide bond between Cys139 and Cys140 was cut
to study the “no-tandem” structures in both chain-A (without substrate)
and chain-B (with substrate). The CO group of Cys139 was replaced
by a hydrogen, meanwhile a hydrogen was added to the -NH group of
Cys140 to fill the open valence.
OLYP functional. OLYP is the combination of Handy’s optimized
exchange (OPTX)31 and LYP correlation.32 Swart et al. have tested
different functionals in calculating the atomization energies for the G2set of up to 148 molecules, six reaction barriers of SN2 reactions,
geometry optimizations of 19 small molecules and 4 metallocenes,
and zero-point vibrational energies for 13 small molecules.33 Their
examination shows that the OPTX containing functionals perform
better than the regular general gradient approximation functionals
(GGAs) like PBE,34,35 BLYP,32,36 and BP.3638 For organic systems,
OLYP has been shown to function as well as the hybrid functional
B3LYP.33,39 Very recently, Hopmann et al. reported that for 57Fe
M€ossbauer isomer shift calculations, the OLYP potential performs
comparably well for iron nitrosyls and for iron complexes in general.40
The resting state of the [4Fe-4S] core in APSR has þ2 charge,
[Fe4S4]2þ. The core plus the four Cys side chains has a net charge of
2, [Fe4S4(Sγ-Cys)4]2. Therefore each of the four equivalent iron sites
has a 2.5þ oxidation state.19,41 The antiferromagnetically (AF) coupled
Stotal = 0 ground state requires two mixed valence iron pairs with opposite
spins. The combinations of the spin states can be: {Fe1VFe2VFe3vFe4v},
{Fe1vFe2VFe3VFe4v}, and {Fe1vFe2VFe3vFe4V}, where “v” and “V” represent
spin up and down, respectively. As in previous work, we perform “brokensymmetry” (BS)4244 calculations to represent the AF-coupled spin
states. First we construct a ferromagnetically (F) spin-coupled (Stotal =
18/2) determinant, where the spins on the four irons are aligned in a
parallel fashion. Then we rotate the spin vector located on two of the iron
sites by interchanging the R and β fit density blocks on the two sites from
the output file TAPE21 created by this F-coupled calculation in ADF to
get the starting spin density for the Stotal = 0 state. These BS states are not
pure Stotal = 0 states. Instead, these states (and their energies) are weighted
averages of the pure spin states, strongly weighted toward the lower Stotal
states based on the spin coupling algebra.19,4144 We have not included
spin projection corrections in the current work since we have estimated
that these will make very small differences in the DFT calculated relative
energies of different states.
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4.1. Geometry Optimization. To determine which one of the
three spin states of the [4Fe-4S] cluster in APSR has the lowest energy
and is geometrically closest to the X-ray crystal structure, we geometry
optimized the active site clusters taken from chain-A (without
substrate binding) and chain-B (with substrate binding) in the
{Fe1VFe2VFe3vFe4v}, {Fe1vFe2VFe3VFe4v}, and {Fe1vFe2VFe3vFe4V}
three BS spin states. All calculations were performed within the
conductor-like screening (COSMO)4548 solvation model with dielectric constant ε = 20. In COSMO, the quantum cluster is embedded
in a molecular shaped cavity surrounded by a continuum dielectric
medium. There is no universal dielectric constant for COSMO-like
solvation calculations. Although the dielectric value ε = 4 is commonly
used for the protein interior, since this is the value for the dielectric
constants of crystalline and polymeric amides49 and dry protein and
peptide powders,5053 many studies show that higher effective dielectric
constant values (430) for protein interiors are needed in reproducing
the pKa values of certain internal ionizable groups.5360 For the current
study, since many charged groups are in the quantum cluster, a larger
dielectric constant (ε = 20) was chosen for the COSMO calculations.
The van der Waals radii for atoms Fe, C, S, P, O, N, and H were taken as
1.5, 1.7, 1.8, 1.8, 1.4, 1.55, and 1.2 Å, respectively.61,62 The probe radius
for the contact surface between the cluster and the solvent was set to
2.0 Å. The triple-ζ (TZP) (for Fe and S) and double-ζ (DZP) (for other
atoms) polarization Slater-type basis sets with frozen cores (C(1s),
N(1s), O(1s), S(1s,2s,2p), P(1s,2s,2p), and Fe(1s,2s,2p) are frozen)
were applied for geometry optimizations. To partially apply the strain
from the protein environment, CR atoms on Lys144, Thr232, Arg245,
and Trp246, Cγ of Arg242, and the link-H atoms on Thr87, Gly88,
His136, Gly137, Cys139, Gly141, Ile142, Lys144, Cys228, Glu229,
Pro230, Thr232, Arg242, Arg245, and Trp246, were fixed during
geometry optimizations. The broken-symmetry state energies obtained
after COSMO geometry optimizations were used to compare the
relative energies in Section 6.
€ ssbauer Isomer Shift and Quadrupole Splitting
4.2. Mo
Calculations. For all models, we applied single-point M€ossbauer
isomer shift and quadruple splitting calculations at the optimized
geometries using all-electron (i.e., without frozen core approximation)
all TZP Slater-type basis sets. First, a high-spin F-coupled single-point
energy calculation was performed at the BS optimized geometry. Its
TAPE21 file was then modified accordingly by interchanging the R and
β fit density blocks on two of the iron sites. Starting from the modified
TAPE21, a BS state single-point energy calculation in COSMO again
with all-electron TZP Slater-type basis sets was performed to obtain the
electron density (F(0)) and the electric field gradient (EFG) at the Fe
nucleus.
The M€ossbauer isomer shifts δ were calculated based on F(0):
δ ¼ RðFð0Þ AÞ þ C
ð1Þ
where A = 11877 is a constant. Our previous isomer shift fit based on 19
Fe2.5þ,3þ,3.5þ,4þ complexes (30 distinct iron sites) using OLYP/
all-electron-TZP method (with INTEGRATION = 4.0) yielded
R = 0.307 and C = 0.385 mm s1.63 The numerical integration
accuracy parameter INTEGRATION = 4.0 is also used for the current
calculations.
For calculating the M€ossbauer quadrupole splittings (ΔEQ), the EFG
tensors V were diagonalized and the eigenvalues were reordered so that
|Vzz| g |Vxx| g |Vyy|. The asymmetry parameter η is defined as
η ¼ jðVXX VYY Þ=Vzz j
ð2Þ
Then the ΔEQ for 57Fe of the nuclear excited state (I = 3/2) can be
calculated as
ΔEQ ¼ ð1=2ÞeQVzz ð1 þ η2 =3Þ1=2
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where e is the electrical charge of a positive electron, Q is the nuclear
quadrupole moment of Fe. We had used eQ = 0.15 electron-barn64 in our
previous publications. For the current study, we applied a slightly
different eQ = 0.158 electron-barn taken from the careful quantum
chemical calculations (nonrelativistic) by Neese’s group.65
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Table 1. Extended X-ray Absorption Fine Structure (EXAFS)
Curve-Fitting Parameters for Fe K-Edgea
4.3. Fitting Atomic Charges from Electrostatic Potentials
(ESP). A modified version of the CHELPG code41,66 was used to fit the
atomic point charges from the molecular electrostatic potentials (ESP)
obtained from the final all-electron all-TZP single-point energy calculations. The singular value decomposition (SVD) method41 was introduced into the code to minimize the uncertainties in the fitting
procedure. The total net charge and the three Cartesian dipole moment
components of each cluster were used as constraint conditions for the fit.
The fitted points lay on a cubic grid between the van der Waals radius
and the outer atomic radius with a grid spacing of 0.2 Å. The outer
atomic radius (5.0 Å here for all atoms) defines the outer boundary of the
electrostatic potential that was used in the charge fitting. The same van
der Waals radii were used as in COSMO calculations.
5. X-RAY ABSORPTION SPECTROSCOPY (XAS) RESULTS AND DISCUSSION
To probe the coordination and geometry of the [4Fe-4S]
cluster in APSR we examined Mt-APSR by XAS. To facilitate
EXAFS analyses, monomeric Mt-APSR was used instead of PaAPSR since Pa-APSR purifies as a tetramer. To determine the
effect of APS binding on Fe-site structure, Mt-APSR was
incubated with a 3-fold stoichiometric excess of APS, at a
concentration exceeding the kd of APS and AMP (0.2 μM and
5.4 μM, respectively9). Therefore, the form of Mt-APSR in
EXAFS experiments was the S-sulfocysteine state with AMP
bound and the C-terminal docked in the active site (closed state).
By contrast, for crystallization of Pa-APSR, ∼60-fold excess of
APS over enzyme was added and the stable form that crystallized
was formed with the C-terminal tail out of the active site (open
state) (see Figure 6C in ref 13).
5.1. Fe K-Edge XAS. The normalized Fe K-edge XANES
spectrum of APSR is shown in Figure 3. This region of the XAS
spectra (1s f 3d transitions) provides valuable information
about metal ion coordination number and oxidation state.67 The
Fe pre-edge area calculated for APSR (∼18 102 eV) is
consistent with a four-coordinate Fe species. This conclusion is
supported by M€
ossbauer studies for native Mt-APSR which
confirm the presence of a [4Fe-4S]2þ cluster.18 The pre-edge
feature observed at ∼7112 eV occurs because of an electronic
dipole-forbidden quadrupole-allowed transition from the Fe 1s
orbital to valence orbitals with significant Fe 3d character.67,68
The increase in intensity of this formally forbidden transition is a
result of mixing of Fe 4p-3d orbitals caused by deviation of the
absorbing Fe center from centrosymmetry.69 To investigate
changes in cluster geometry and coordination during catalysis,
Mt-APSR was incubated with APS to form the S-sulfocysteine
intermediate with AMP bound in the active site and XAS was
recorded for this stable conformation of the enzyme (Table 1,
Supporting Information, Figure 2). No shift in position or
intensity of the pre-edge energy was observed for the Mt-APSR
S-sulfocysteine intermediate, indicating that the level of 4p
mixing is identical to native Mt-APSR. Since the 1s f 3d
transition is very sensitive to local Fe-site structure, this is a
sensitive indication that not only is there no change in oxidation
state or coordination number, but there is unlikely to be more
than a very small change in geometry of the Fe in the [4Fe-4S]
interaction
N
R (Å)
σ2 103
F
Mt-APSR
FeS
4*
2.297
3.9
1.93
sample 1
FeFe
FeS
3*
3.9
2.726
2.297
2.4
3.8
1.92
FeFe
2.1
2.726
2.7
Mt-APSR
FeS
4*
2.294
3.9
sample 2
FeFe
3*
2.729
2.0
sample
1.92
FeS
3.7
2.294
3.4
FeFe
1.7
2.729
1.3
Mt-APSR þ APSb
FeS
4*
2.298
3.9
1.28
sample 1
FeFe
FeS
3*
3.8
2.728
2.298
2.2
3.6
0.93
FeFe
2.0
2.728
2.2
Mt-APSR þ APSb
FeS
4*
2.297
3.8
sample 2
FeFe
3*
2.730
2.5
FeS
3.6
2.297
3.4
FeFe
1.8
2.730
2.0
1.86
1.48
1.44
a
Coordination Number (N), Interatomic Distances (R), Mean-Square
Deviations in Interatomic Distance (σ2, Å2), Fit-Error Function (F) is
Defined as Æ{Σk6 (χcalc* χexpt)2/Σχ2expt}1/2æ. For Mt-APSR the k
Range was 2.3517 (Filtered R = 1.23); for Mt-APSR þ APS the k
Range was 2.1517 (Filtered R = 1.23); giving resolution ∼0. 1 Å and
∼17 independent degrees of freedom. Fits are shown both for fixed
(marked with *) and variable N. b The enzyme was in the S-sulfocysteine
state with AMP bound and the C-terminal docked in the active site.
cluster between the native and S-sulfocysteine intermediate
forms of Mt-APSR.
5.2. EXAFS. The EXAFS data for Mt-APSR, reported at highresolution to k ∼ 17 Å1, is shown in Figure 3 (inset). The
pattern in the data reflects the fact that there are at least two shells
of scatterers, sulfur and iron, significantly contributing to the
EXAFS spectrum. The Fourier transform (FT) of Mt-APSR
(Figure 3, Table 1) shows an intense first-shell FeS interaction
at 2.3 Å and a slightly less intense second-shell FeFe interaction
at 2.73 Å. For this k range, resolution in R space is ∼0.1 Å,
meaning that if the FeFe or FeS distances differed by >0.1 Å
they should, in principle, be resolvable, and that if the distances
differ by > ∼0.12 Å they should be readily resolvable into two
different shells of scatterers (Two FeFe distances that differed
by 0.12 Å would show a “beat” in the EXAFS at ∼k = 13 Å1
which would be readily detectable). No improvement was seen
for multiple shell fits. It is possible, of course, that there are not
two, but up to six different FeFe distances (Note that if there
were significant disorder, with multiple forms of the Fe/S cluster,
this could increase further the number of FeFe distances. This
is ruled out by the absence of disorder in the Mossbauer spectra).
In this case, individual FeFe distances might not be resolvable.
However, if this were the case, there would be an increase in the
DebyeWaller factor. However, the observed DebyeWaller
factors are small, indicating that there is, at most a very small
(<0.12 Å) spread in FeFe distance, consistent with the spread
in FeFe distances that is found in the DFT calculations
(Tables 2 and 3).
If the experimental FeFe σ2 value of ∼2.5 103 Å2 is
attributed solely to static disorder and if the most asymmetric
possible distribution of FeFe distances is assumed (i.e., 5
short and 1 long distance), the spread in FeFe distances is
6616
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Inorganic Chemistry
ARTICLE
Table 2. FeFe and Fe-ligand Bond Lengths (Å), Broken-Symmetry State Energies (E, eV), Fe Net Spin Populations (NSP), and
M€
ossbauer Properties (Isomer shifts δ, Quadrupole Splittings ΔEQ, mm s1, and η) of the Resting State [Fe4S4(Sγ-Cys)4]2
Cluster in Wild-Type Pa-APSR without Substrate Binding: Comparison between Calculations and Experiments (Exp)
calculated chain-A without APS
V
V
v
Fe1 Fe2 Fe3 Fe4
a
v
Fe1vFe2VFe3VFe4v
Fe1vFe2VFe3vFe4V
expa
Fe1Fe2
2.685
2.700
2.651
2.7
Fe1Fe3
2.657
2.687
2.662
2.7
Fe1Fe4
2.707
2.742
2.724
2.7
Fe2Fe3
2.758
2.963
2.767
2.7
Fe2Fe4
Fe3Fe4
2.684
2.646
2.711
2.671
2.750
2.663
2.8
2.8
Fe1S1
2.309
2.219
2.291
2.2
Fe1S2
2.323
2.305
2.222
2.2
Fe1S3
2.232
2.299
2.300
2.2
Fe2S1
2.339
2.349
2.259
2.3
Fe2S2
2.293
2.191
2.290
2.3
Fe2S4
2.269
2.341
2.335
2.2
Fe3S1
Fe3S3
2.211
2.313
2.293
2.229
2.286
2.309
2.3
2.3
Fe3S4
2.330
2.345
2.255
2.3
Fe4S2
2.201
2.267
2.287
2.3
Fe4S3
2.314
2.306
2.238
2.3
Fe4S4
2.322
2.247
2.321
2.3
Fe1Sγ-Cys140
2.249
2.270
2.333
2.3
Fe2Sγ-Cys139
2.311
2.312
2.267
2.3
Fe3Sγ-Cys228
Fe4Sγ-Cys231
2.301
2.277
2.325
2.298
2.292
2.324
2.3
2.3
E
1193.735
1193.369
1193.532
NSP(Fe1, Fe2, Fe3, Fe4)
(3.11, 3.18, 3.16, 3.11)
(3.12, 3.17, 3.17, 3.14)
(3.10, 3.18, 3.20, 3.15)
δ(Fe1, Fe2, Fe3, Fe4)
(0.43, 0.48, 0.45, 0.44)
(0.42, 0.46, 0.46, 0.44)
(0.39. 0.46, 0.43, 0.43)
average δ
0.45
0.45
0.43
ΔEQ(Fe1, Fe2, Fe3, Fe4)
(1.17, 1.01, 0.80, 1.01)
(0.95, 1.20, 1.20, 0.78)
(0.91, 0.99, 1.04, 1.32)
average |ΔEQ|
1.00
1.03
1.07
η(Fe1, Fe2, Fe3, Fe4)
(0.48, 0.62, 0.65, 0.92)
(1.00, 0.31, 0.24, 0.77)
(0.96, 0.46, 0.55, 0.35)
0.4518
1.0918
The crystal structure of Pa-APSR was at 2.7 Å resolution, PDB code: 2GOY.13 Distances are taken from chain-A without substrate binding.
√
∼ 6σ = 0.12 Å. A more reasonable estimate would recognize
that the experimental σ2 is the sum of σ2vib and σ2static, The
former can be estimated from the fitted σ2 value for Pyrococcus
furiousus ferredoxin, 2.0 103 Å2. If this is attributed to
vibrational motion, the apparent σ2static is 5.0 104 Å2,
suggesting a spread in FeFe distances of ∼0.05 Å. While the
true spread in FeFe distances will depend on details of the
distribution in FeFe distances and the vibrational contribution
to σ2, it should be between these limits.
Coordination numbers were initially constrained to the chemically correct values (4 FeS and 3 FeFe). These gave
excellent fits. Modest improvement in fit quality was possible if
the coordination number was allowed to vary; this improvement
is consistent with that expected for a 50% increase in variable
parameters. To confirm that these data are consistent with a
[4Fe-4S] cluster, we compared the EXAFS and FT of Mt-APSR
with those previously reported data for P. furiosus ferredoxin
containing either an [3Fe-4S] cluster or a [4Fe-4S] clusters (data
provided by Prof. G.N. George, University of Saskatoon). Supporting
Information, Figure 3 unambiguously shows that the EXAFS
spectrum for Mt-APSR is in good agreement with the spectrum
for the [4Fe-4S] cluster in ferredoxin, albeit with a slightly less
intense FeFe peak, suggesting somewhat larger disorder in the
FeFe scattering in Mt-APSR, consistent with the fitting results.
The EXAFS spectrum and FT of the Mt-APSR S-sulfocysteine
intermediate are identical to those of native Mt-APSR, and the
FeS and FeFe bond lengths remain consistent (Table 1,
Supporting Information, Figures 2B and 2C). Although the
accuracy of EXAFS bond-lengths is generally taken as ∼0.02 Å,
the precision is much better. We have found previously that the
precision of biological EXAFS data is ∼0.003 Å2;24 this is
reflected in the excellent reproducibility of the duplicate samples
in Table 1. This precision, together with the invariance seen in
Table 1, indicates that the average core structure of the [4Fe4S] cluster in Mt-APSR is unaffected in the S-sulfocysteine
conformation of the protein. Since XAS spectra reflect bondlengths that are averaged over all the atoms in the cluster, it is in
principle possible that small changes at one site could be
compensated by equal but opposite changes at another site,
such that the average distances remain the same. To probe these
changes at high resolution, density functional theory calculations were undertaken.
6617
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Inorganic Chemistry
ARTICLE
Table 3. FeFe and Fe-ligand Bond Lengths (Å), Broken-Symmetry State Energies (E, eV), Fe Net Spin Populations (NSP), and
M€
ossbauer Properties (Isomer shifts δ, Quadrupole Splittings ΔEQ, mm s1, and η) of the Resting State [Fe4S4(Sγ-Cys)4]2
Cluster in Wild-Type Pa-APSR with Substrate Binding: Comparison between Calculations and Experiments (Exp)
calculated chain-B with APS
V
V
v
v
Fe1vFe2VFe3VFe4v
Fe1 Fe2 Fe3 Fe4
a
Fe1vFe2VFe3vFe4V
expa
Fe1Fe2
2.741
2.718
2.669
2.7
Fe1Fe3
2.683
2.690
2.717
2.7
Fe1Fe4
2.727
2.813
2.742
2.7
Fe2Fe3
2.732
2.863
2.750
2.7
Fe2Fe4
Fe3Fe4
2.680
2.728
2.708
2.670
2.792
2.678
2.7
2.7
2.3
Fe1S1
2.304
2.205
2.303
Fe1S2
2.333
2.308
2.227
2.3
Fe1S3
2.209
2.310
2.314
2.3
Fe2S1
2.313
2.308
2.221
2.3
Fe2S2
2.267
2.183
2.274
2.2
Fe2S4
2.247
2.343
2.338
2.3
Fe3S1
Fe3S3
2.178
2.310
2.267
2.222
2.271
2.308
2.2
2.2
Fe3S4
2.305
2.293
2.217
2.2
Fe4S2
2.193
2.273
2.285
2.3
Fe4S3
2.290
2.284
2.202
2.3
Fe4S4
2.310
2.230
2.302
2.3
Fe1Sγ-Cys140
2.304
2.329
2.326
2.3
Fe2Sγ-Cys139
2.293
2.325
2.315
2.3
Fe3Sγ-Cys228
Fe4Sγ-Cys231
2.290
2.257
2.306
2.289
2.306
2.276
2.3
2.3
E
1446.545
1446.291
1446.501
NSP(Fe1, Fe2, Fe3, Fe4)
(3.13, 3.11, 3.12, 3.07)
(3.14, 3.16, 3.12, 3.13)
(3.14, 3.14, 3.17, 3.13)
δ(Fe1, Fe2, Fe3, Fe4)
(0.45, 0.45, 0.43, 0.42)
(0.46, 0.45, 0.43, 0.43)
(0.47, 0.46, 0.44, 0.43)
average δ
0.44
0.44
0.45
ΔEQ(Fe1, Fe2, Fe3, Fe4)
(1.08, 0.95, 0.79, 1.03)
(0.87, 1.24, 1.15, 0.70)
(0.85, 1.04, 0.87, 1.23)
average |ΔEQ|
0.96
0.99
1.00
η(Fe1, Fe2, Fe3, Fe4)
(0.33, 0.65, 0.53, 0.88)
(0.78, 0.34, 0.29, 0.62)
(0.89, 0.64, 0.42, 0.33)
0.4518
1.1218
The crystal structure was at 2.7 Å resolution, PDB code: 2GOY.13 Distances are taken from chain-B with substrate binding.
6. DFT CALCULATION RESULTS AND DISCUSSION
6.1. Calculating Results for Wild-Type [Fe4S4(Sγ-Cys)4]2
Models. The main calculated properties for different spin
states of the wild-type [4Fe-4S] cluster without and with the
substrate are given in Tables 2 and 3, respectively. The
calculated FeFe and FeS distances and 57Fe M€ossbauer
isomer shifts and quadrupole splittings are compared with
available experimental results.18
As previously reported19,41 each of the optimized [Fe4S4(SγCys)4]2 clusters has a compression structure with four “short”
(∼2.2 Å) and eight “long” (∼2.3 Å) FeS distances. The two
irons with the same spin direction are in an Fe2S2 rhomb
distorted butterfly “plane” with four “long” FeS distances,
and the four FeS bonds between the two “planes” have “short”
distances. Taking the {Fe1VFe2VFe3vFe4v} state in Table 2
(without APS binding) as an example, Fe1 and Fe2 (spin down)
are on the plane containing Fe1S1Fe2S2, and Fe3 and Fe4
(spin up) are on the plane with Fe3S3Fe4S4. As a result,
the eight FeS bond lengths on the two planes are long (in
average 2.32 Å), and the four Fe1S3, Fe2S4, Fe3S1, and
Fe4S2 bonds between the two planes are shorter (in average
2.23 Å). Overall, the spread of the DFT calculated FeS
(2.202.34 Å) and FeFe (2.652.76 Å) distances agree with
the EXAFS’s results of ∼2.3 ( 0.1 Å and ∼2.7 ( 0.1 Å,
respectively. Since the Pa-APSR crystal structure was solved at
2.7 Å resolution,13 the compression structure for the [4Fe-4S]
cluster is not obvious in either chain-A (without APS binding) or
chain-B (with APS binding). It is also not reliable to determine
the spin state of the cluster based on the comparison between the
calculated geometries and the crystal structure. Since the
{Fe1VFe2VFe3vFe4v} state yields the lowest broken-symmetry
energy in both chain-A and chain-B, it is likely that the
[Fe4S4(Sγ-Cys)4]2 cluster of Pa-APSR is in the {Fe1VFe2VFe3vFe4v}
state before and after substrate binding.
In the presence of APS, the calculated distances of Fe1Fe2
and Fe3Fe4 are increased by 0.06 Å; the four “short” distances
(Fe1S3, Fe2S4, Fe3S1, and Fe4S2) between the two
planes “Fe1S1Fe2S2” and “Fe3S3Fe4S4” are shortened by 0.02 Å on average. Since the thiolate of Cys140 now has a
direct H-bonding interaction with the Lys144 side chain
(Figure 6), the Fe1Sγ-Cys140 bond is increased by 0.06 Å,
6618
dx.doi.org/10.1021/ic200446c |Inorg. Chem. 2011, 50, 6610–6625
Inorganic Chemistry
ARTICLE
Table 4. Comparing the Calculated ESP Atomic Charges of
the [Fe4S4(Sγ-Cys)4]2 Quantum Cluster in the Fe1VFe2VFe3vFe4v Spin State in Different Models
chain-A without APS
wild-
no-
type
tandem
K144A
chain-B with APS
wild-
no-
type
tandem
K144A
Table 5. Calculated FeFe and Fe-ligand Bond Lengths (Å)
and Fe Net Spin Populations (NSP) for the [Fe4S4(SγCys)4]2 Quantum Cluster in Fe1VFe2VFe3vFe4v Spin State
with Substrate and without Substrate Binding in No-tandem
and K144A Models
chain-A without APS
chain-B with APS
no-tandem
K144A
no-tandem
K144A
Fe1Fe2
2.755
2.710
2.819
2.720
Fe1Fe3
Fe1Fe4
2.683
2.745
2.661
2.716
2.700
2.761
2.676
2.733
Fe2Fe3
2.742
2.754
2.735
2.731
Fe2Fe4
2.680
2.694
2.649
2.696
0.54
Fe3Fe4
2.662
2.689
2.705
2.727
0.67
0.71
Fe1S1
2.317
2.311
2.306
2.303
0.45
0.39
0.59
Fe1S2
2.340
2.323
2.326
2.317
0.59
0.64
0.67
0.66
Fe1S3
2.238
2.224
2.218
2.208
0.60
0.66
0.70
0.70
0.70
0.67
2.15
0.67
2.28
0.63
2.19
0.66
2.22
0.68
2.44
Fe2S1
Fe2S2
2.349
2.284
2.340
2.298
2.311
2.272
2.317
2.276
Fe2S4
2.253
2.262
2.242
2.251
Fe3S1
2.218
2.203
2.210
2.175
Fe3S3
2.325
2.340
2.308
2.317
Fe3S4
2.333
2.340
2.298
2.302
Fe4S2
2.208
2.203
2.199
2.201
Fe4S3
2.326
2.335
2.296
2.296
Fe4S4
Fe1Sγ-Cys140
2.332
2.279
2.341
2.254
2.310
2.323
2.318
2.270
Fe2Sγ-Cys139
2.303
2.313
2.281
2.298
Fe3Sγ-Cys228
2.325
2.303
2.293
2.284
Fe4Sγ-Cys231
2.288
2.284
2.257
2.276
rmsd a
0.022
0.014
0.022
0.012
NSP(Fe1)
3.15
3.12
3.13
3.11
NSP(Fe2)
3.16
3.19
3.11
3.13
NSP(Fe3)
NSP(Fe4)
3.18
3.14
3.18
3.14
3.12
3.07
3.12
3.10
Fe1
0.43
0.43
0.47
0.45
0.42
0.50
Fe2
0.58
0.58
0.60
0.61
0.59
0.61
Fe3
0.62
0.63
0.63
0.61
0.65
0.61
Fe4
S1
0.62
0.44
0.60
0.46
0.63
0.48
0.62
0.44
0.61
0.48
0.64
0.46
S2
0.45
0.44
0.44
0.44
0.43
0.46
S3
0.47
0.46
0.53
0.48
0.49
S4
0.70
0.68
0.70
0.70
Sγ-Cys140
0.41
0.40
0.54
Sγ-Cys139
0.63
0.68
Sγ-Cys228
0.61
Sγ-Cys231
∑total
0.65
2.11
and it becomes the longest among the four FeSγ-Cys bonds.
The elongation of the Fe1Sγ-Cys140 distance may explain the
marked change in FeSγ stretching modes observed in the
Resonance Raman spectra of Pa-APSR upon APS binding.17
The net spin populations (NSP) from Mulliken population
analysis are the main indication of the high-spin or intermediatespin character of the metal sites. In the ideal ionic limit, the net
unpaired spin populations are 5 and 4 for the high-spin Fe3þ (five
d-electrons) and Fe2þ (six d-electrons) sites, respectively. Therefore, for the delocalized spins between the high-spin Fe3þ and
Fe2þ sites, one should expect the average net spin of 4.5.
However, because of the Fe-ligand covalency, our previous
calculations show that the calculated net spin magnitude for a
high-spin Fe3þ/Fe2þ site is normally by about 1 e smaller than
the ionic limit.19,41,7073 The current calculated net spins on the
four Fe sites in both chain-A and chain-B are 3.13.2 (Tables 2
and 3), about 1.31.4 e smaller than 4.5, indicative of the high
spin Fe2.5þ sites with substantial FeS covalency. The opposite
signs for the spin densities indicate the AF-coupling.
Experimentally derived 57Fe M€ossbauer parameters were almost identical for the [Fe4S4(Sγ-Cys)4]2 cluster for native APSR
and for the S-sulfocysteine intermediate form with AMP bound
and C-terminal tail docked in the active site. Note that the
M€ossbauer analyses on Mt-APSR18 were performed by incubating
the enzyme with a 2-fold stoichiometric excess of APS at a
concentration well above the Kd of APS or AMP, similar to the
current EXAFS study. Our calculations show the subtle differences
of the M€ossbauer isomer shifts and quadrupole splittings for each
Fe site in different spin states, with or without substrate. However
the predicted isomer shifts are all around 0.45 mm s1, in excellent
agreement with the experiment. The average predicted quadrupole
splitting values (∼1 mm s1) also agree well with the experimental
values (Tables 2 and 3).
To understand how APS binding influences the charge
distributions of the [4Fe-4S] cluster, we calculated the ESP
atomic charges of the [Fe4S4(Sγ-Cys)4]2 clusters in chain-A
and chain-B in the Fe1VFe2VFe3vFe4v spin state. The ESP charges
are given in Table 4 under the “wild-type” columns.
a
Root mean squares difference of the calculated FeFe and FeS
distances between the current modified models and the optimized wildtype models (in Fe1VFe2VFe3vFe4v state) given in Table 2 (for chain-A)
and Table 3 (for chain-B).
In chain-A without APS, Fe1 has the least positive charge
(0.43), which is approximately 0.2 charge units less than Fe3 and
Fe4. In addition, the Cys140 thiolate which coordinates to Fe1
has the least negative charge (0.41), which is also by 0.2 charge
units in magnitude less than the other three Cys residues. This
shows the charge transfer from the Cys140 thiolate to the nearby
cationic group of Lys144 side chain. In chain-B (Figure 6),
Lys144 is in between Cys140 and APS. The distances between
Sγ-Cys140 and N-Lys144, and between N-Lys144 and O-APS
are 3.16 Å (3.56 Å) and 3.19 Å (3.03 Å), respectively, in the X-ray
(DFT optimized) structure of chain-B. The charge transfer from
Cys140 to Lys144 is decreased in the presence of APS. As a result,
Sγ-Cys140 becomes more negative from 0.41 (chain-A) to
0.45 (chain-B), and the ESP charge on Fe1 is slightly increased
from 0.43 (chain-A) to 0.45 (chain-B). The repulsion between
APS and Cys228 also increases the magnitude of the negative
charge on Sγ-Cys228 from 0.61 (chain-A) to 0.70 (chain-B).
These changes result in a subtle increase in the total negative ESP
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Inorganic Chemistry
charge of the entire [Fe4S4(Sγ-Cys)4]2 cluster from 2.11
(Chain-A) to 2.19 (Chain-B).
Even though APS does not have a direct interaction with the
[4Fe-4S] cluster, the midrange electrostatic interactions between
the two species indeed influence the charge distribution and the
detailed geometry within the [4Fe-4S] cluster, although the
geometric changes are not large enough to be observed in EXAFS
and M€ossbauer experiments.
6.2. Calculating Results for No-Tandem [Fe4S4(Sγ-Cys)4]2
Models. The main FeFe and FeS distances of the optimized
no-tandem models in the {Fe1VFe2VFe3vFe4v} state with and
without APS are shown in Table 5, in the columns under “notandem”. After breaking the peptide bond between Cys139 and
Cys140 (Figure 7), the [4Fe-4S]2þ core in both chain-A and
chain-B expands. For chain-A without APS, the Fe1Fe2
distance is increased by 0.08 Å compared to wild-type
(Table 2). Fe1Fe3, Fe1Fe4, and Fe3Fe4 are also increased
by 0.03, 0.04, and 0.02 Å, respectively. Except for Fe2S2,
Fe2S4, and Fe2Sγ-Cys139, which are decreased by 0.01 Å, all
other FeS distances are increased by 00.03 Å. The rmsd of
the FeFe and FeS distances between the calculated
no-tandem model and the wild-type optimized cluster of chainA is 0.022 Å. The angles — Fe1Sγ-Cβ-Cys140 and — Fe2SγCβ-Cys139 are widened from 112.6 and 109.6 to 117.3
and 112.4, respectively. Meanwhile the distance between
(Sγ-Cys140) and (Sγ-Cys139) is enlarged from 6.19 Å to 6.43 Å.
From chain-A to chain-B for the no-tandem models, the
Fe1Fe2 distance is further increased by 0.06 Å, and reaches
2.82 Å, which is the longest FeFe distance for the
{Fe1VFe2VFe3vFe4v} state of the [4Fe-4S] cluster in the current
study. For chain-B with APS, the rmsd of the FeFe and FeS
distances between the no-tandem model and the optimized wildtype cluster is also 0.022 Å.
Thus, the unique tandem pair Cys139 and Cys140 in Pa-APSR
keeps the [Fe4S4(Sγ-Cys)4]2 cluster more compact, which
works to prevent the cluster from being reduced. It is known
that, once the [Fe4S4(Sγ-Cys)4]2 cluster is reduced (net charge
of the cluster would be 3), the repulsion among the S 3 3 3 S
atoms would be stronger, forcing the expansion of the overall
cluster.19,41 The tandem structure in Pa-APSR makes the expansion of the [4Fe-4S] energetically unfavorable, which explains why
it is difficult to reduce the cluster.18 After several attempts, we were
able to reduce the [Fe4S4(Sγ-Cys)4]2 cluster in Mt-APSR, but
the reduction efficiency was at most 44% of the total protein.18
The calculated ESP atomic charges for the [Fe4S4(SγCys)4]2 cluster in the no-tandem models in both chain-A and
chain-B are given in Table 4 in the columns under “no-tandem”.
In both chain-A and chain-B, Fe1 still holds the least positive ESP
charge. Overall, the calculated ESP charges of the no-tandem
models are similar to the corresponding wild-type results. The
total ESP charges (∑total) of the no-tandem models are a little
more negative than those of the wild-type cluster, which increases
the possibility of one-electron oxidation of the [4Fe-4S] cluster
in the no-tandem form. The propensity and rate of cluster
degradation after oxidation is a complex multistep problem,
and would be expected to involve both the covalent Cys linkages
and H-bonding interactions with surrounding residues.
The function of the unique tandem pair Cys139 and Cys140 in
Pa-APSR is to protect the [4Fe-4S] cluster from reduction as will
be examined by redox potential calculations in Section 6.4. Note
that our current method to construct the no-tandem models is
the simplest, but may not represent the best approach. An
ARTICLE
alternative possibility would be to insert one or more residues
between the Cys139 and Cys140 pair. However, this would cause
large conformational change and increase the size of the clusters,
which currently contain 211 (in chain-A) and 250 (in chain-B)
atoms, and approach the reasonable limit of DFT/COSMO
calculations. This issue could be examined in future studies,
ostensibly starting with a smaller overall cluster size.
6.3. Calculating Results for K144A [Fe4S4(Sγ-Cys)4]2 Models. The calculated FeFe and FeS distances of the optimized
K144A models in the {Fe1VFe2VFe3vFe4v} spin state with and
without APS binding are shown in Table 5, in the columns under
“K144A”. The calculated ESP atomic charges for the [Fe4S4(SγCys)4]2 portion of the K144A models in both chain-A and chainB have also been shown in Table 4 in the columns under “K144A”.
There is marked anisotropy in the change of charge distribution and bond lengths when Lys144 is mutated to Ala. In
particular, without charge transfer to Lys144, the negative ESP
charge on Sγ-Cys140 changes significantly from 0.41 (and
0.45) in the wild type to 0.54 (and 0.59) in the K144A
model of chain-A (and chain-B) (Table 4). This influences the
charge on Fe1 such that Fe1 is more positive and in turn, the
charge on S3 is more negative in both models of K144A,
compared to the respective wild type model. Overall the total
charge of the [Fe4S4(Sγ-Cys)4]2 cluster becomes more negative
from 2.11 in the wild type to 2.28 in the K144A models for
chain-A, and from 2.19 to 2.44 for chain-B, respectively
(Table 4). The increase of the negative charge of the [Fe4S4(SγCys)4]2 cluster, in combination with the electrostatic interaction of the negatively charged cluster with the Lys144þ versus
neutral Ala144, explains why the FeS cluster is even more
difficult to reduce in K144A mutant as observed in EPR
experiments.18 The reduction potential of the K144A clusters
will also be examined in Section 6.4.
In chain-B, loss of the H-bonding interaction between SγCys140 and Lys144 decreases the Fe1Sγ-Cys140 bond length
by 0.03 Å, which is greater than the other corresponding FeSγCys distance changes. In general, the rmsd of the calculated
FeFe and FeS distances between the K144A model and the
optimized wild-type cluster is 0.014 Å for chain-A and 0.012 Å for
chain-B. The cluster is more compact in the wild type protein
than in the K144A model. For instance, in chain-A without APS
binding, except for Fe2Fe3, Fe1S2, Fe1S3, Fe2S4, and
Fe3S1, which either remain unchanged or are decreased by less
than 0.01 Å, all other FeFe and FeS distances are increased by
00.04 Å from the wild-type cluster to the K144A model.
Overall the cluster in K144A expands compared to the wild-type
optimized cluster, which is consistent with the increasing of the
electron density over the [Fe4S4(Sγ-Cys)4]2 cluster, and the
increasing lability of the cluster in K144A as observed in EPR
experiments.18
Additionally, a comparison between ESP charges for APS in
the wild-type and K144A models shows that the overall charge on
APS becomes more negative changing from 1.75 in wild type to
1.84 in the K144A model (data not shown). As indicated
above, without charge transfer to Lys144þ, the calculated total
ESP charge of the [Fe4S4(Sγ-Cys)4]2 cluster also becomes
more negative from 2.19 in wild-type to 2.44 in K144A
(chain-B). This supports the crucial role of Lys144 such that in
the absence of the positive charge, the H-bonding interactions
are lost and the negative charges on both the cluster and APS are
more localized making the cluster and APS more negative in the
K144A model. This would result in a strong repulsion between
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ARTICLE
Table 6. Calculated FeFe and Fe-Ligand Bond
Lengths (Å) and Fe Net Spin Populations (NSP) for
the [Fe4S4(Sγ-Cys)4]3 Quantum Cluster in
{Fe12þVFe22þVFe32.5þvFe42.5þv} State with Substrate
and without Substrate Binding in Wild-Type, No-tandem
and K144A Models
chain-A without APS
chain-B with APS
wild-type no-tandem K144A wild-type no-tandem K144A
Fe1Fe2
2.704
2.788
2.706
2.755
2.809
2.683
Fe1Fe3
2.661
2.700
2.663
2.676
2.708
2.685
Fe1Fe4
2.694
2.730
2.700
2.731
2.749
2.740
Fe2Fe3
Fe2Fe4
2.761
2.729
2.735
2.718
2.750
2.737
2.746
2.723
2.719
2.705
2.725
2.729
Fe3Fe4
2.635
2.636
2.634
2.676
2.655
2.672
Fe1S1
2.325
2.327
2.321
2.319
2.307
2.306
Fe1S2
2.305
2.321
2.301
2.281
2.301
2.297
Fe1S3
2.270
2.294
2.276
2.263
2.263
2.265
Fe2S1
2.318
2.324
2.317
2.296
2.300
2.291
Fe2S2
2.299
2.295
2.300
2.271
2.276
2.279
Fe2S4
Fe3S1
2.317
2.213
2.292
2.226
2.319
2.206
2.298
2.192
2.274
2.219
2.317
2.184
Fe3S3
2.357
2.348
2.370
2.345
2.340
2.348
Fe3S4
2.373
2.358
2.363
2.323
2.315
2.321
Fe4S2
2.211
2.221
2.211
2.204
2.212
2.203
Fe4S3
2.333
2.332
2.368
2.296
2.300
2.308
Fe4S4
2.364
2.358
2.340
2.331
2.323
2.338
Fe1Sγ-
2.296
2.336
2.296
2.329
2.387
2.313
Cys140
Fe2Sγ-
2.347
2.329
2.339
2.330
2.309
2.324
2.349
2.350
2.351
2.329
2.337
2.318
2.338
2.349
2.331
2.319
2.318
2.322
Cys139
Fe3SγCys228
Fe4SγCys231
NSP(Fe1)
2.93
3.01
2.92
2.92
2.99
2.94
NSP(Fe2)
NSP(Fe3)
3.00
3.26
2.94
3.27
3.01
3.25
2.97
3.23
2.92
3.23
2.94
3.21
NSP(Fe4)
3.22
3.24
3.23
3.19
3.20
3.21
the [Fe4S4(Sγ-Cys)4]2 cluster and APS that is detrimental to
substrate binding in the mutant protein. In fact, kinetic studies
show that there is a 400-fold decrease in the Kd of APS for
Lys144Ala Mt-APSR and consequently, a decrease in the catalytic efficiency of the mutant protein by almost 63,000-fold as
compared to wild type Mt-APSR.18
Compared to wild-type chain-B, the calculated distances of
(S-APS) 3 3 3 (Sγ-Cys140), (S-APS) 3 3 3 Fe1, and (S-APS) 3 3 3 Fe2
increase from 6.79, 8.20, and 10.84 Å, respectively in the wild
type to 7.29, 8.57, and 11.15 Å, respectively in the K144A model.
Therefore, without Lys144 bridging between APS and Cys140,
repulsion causes APS and the [Fe4S4(Sγ-Cys)4]2 cluster to
move away from each other.
6.4. Calculated [Fe4S4(Sγ-Cys)4]3 Clusters and the Reduction potentials of [Fe4S4(Sγ-Cys)4]2 þ e f [Fe4S4(SγCys)4]3 in Different Models. If an electron is transferred to the
resting state of the [4Fe-4S]2þ core in APSR, the core will change
to [4Fe-4S]1þ, and the core plus the four Cys side chains will
have a net charge of 3, [Fe4S4(Sγ-Cys)4]3. Since the [4Fe4S]2þ core is likely to be in the {Fe1VFe2VFe3vFe4v} spin state,
the added electron then either goes to the pair {Fe1,Fe2} to form
the {Fe12þVFe22þVFe32.5þvFe42.5þv} state, or goes to (Fe3,Fe4}
to form the {Fe12.5þVFe22.5þVFe32þvFe42þv} state. Our geometry optimizations show that these two states yield very similar
broken-symmetry energies (less than 2 kcal mol1 difference) for
all the cluster models. And for most of the models (only wild-type
chain-A is an exception), the {Fe12þVFe22þVFe32.5þvFe42.5þv}
state is slightly lower in energy than the corresponding
{Fe12.5þVFe22.5þVFe32þvFe42þv} state. Therefore, here we only
present the geometries (Tables 6 and 7) and energies (Table 7) of
the {Fe12þVFe22þVFe32.5þvFe42.5þv} state for all the cluster models.
The formal net spin population of a high-spin Fe2þ ion is 4.
Our calculated magnitudes of the net spins (2.923.01) of
Fe1 and Fe2 in all the {Fe12þVFe22þVFe32.5þvFe42.5þv} state
[Fe4S4(Sγ-Cys)4]3 clusters are about 1 e smaller than 4
(Table 6). The calculated net spin values for Fe3 and Fe4 in
the [Fe4S4(Sγ-Cys)4]3 clusters (Table 6) are about 0.1 larger
than the corresponding ones in the [Fe4S4(Sγ-Cys)4]2 clusters
(Tables 2, 3, and 5). One measure of the metalligand covalency
is the ratio of the calculated to the formal site spin population
(spin population ratio), and lower percentages represent greater
covalency. In wild-type chain-A, the spin population ratios of the
four iron sites change from (69%, 71%, 70%, 69%) in the
{Fe12.5þVFe22.5þVFe32.5þvFe42.5þv} state [Fe4S4(Sγ-Cys)4]2
cluster to (73%, 75%, 72%, 72%) in the {Fe12þVFe22þVFe32.5þvFe42.5þv} state [Fe4S4(Sγ-Cys)4]3 cluster. Very similar
percentage changes are also obtained for other model clusters.
Therefore, with an additional electron, the [4Fe-4S]1þ core of
the [Fe4S4(Sγ-Cys)4]3 cluster in general has less covalency
(larger spin population ratio) than the [4Fe-4S]2þ core of the
[Fe4S4(Sγ-Cys)4]2 cluster. As a result, upon 1e reduction, the
[4Fe-4S] core expands. This is seen relatively easier by comparing the average (avg) FeFe and FeS distances (Table 7).
Especially for the wild-type chain-A model, where upon 1e
reduction, the (FeFe)avg elongates from 2.690 Å to 2.697 Å, the
average of the four FeS distances of Fe1S3, Fe2S4,
Fe3S1, and Fe4S2 is increased from 2.228 Å to 2.253 Å,
and the average distance of the eight FeS bonds on the
“Fe1S1Fe2S2” and “Fe3S3Fe4S4” planes is also
increased from 2.318 Å to 2.334 Å.
On the basis of the OLYP/COSMO calculated brokensymmetry state energies (E), we have calculated the reduction
potentials (E0) of [Fe4S4(Sγ-Cys)4]2 þ e f [Fe4S4(SγCys)4]3 for all the cluster models according to
E0 ¼ Ef½Fe4 S4 ðSγ -CysÞ4 2 g Ef½Fe4 S4 ðSγ -CysÞ4 3 g þ ΔSHE
ð4Þ
where ΔSHE is the standard hydrogen electrode potential. Here
we use ΔSHE = 4.34 V, obtained from Lewis et al.’s calculations based solely on experimental data plus our electron energy
threshold correction.7476 The calculated reduction potentials
for all the model clusters are given in Table 7.
The measured 2/3 reduction potentials of the [4Fe-4S]
clusters in ferredoxins are from 0.28 to 0.45 V.7779 So far,
reduction of the [Fe4S4(Sγ-Cys)4]2 cluster in Pa-APSR has not
been successful. In our recent EPR experiment, we could photo
reduce the [Fe4S4(Sγ-Cys)4]2 cluster in Mt-APSR in the presence of deazaflavin/oxalate with at most 44% reduction
efficiency.18 It is likely that the 2/3 reduction potential of
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ARTICLE
Table 7. Averages (avg) of the Calculated FeFe and Fe-Ligand Bond Lengths (Å), Broken-Symmetry State Energies (E, eV), and
Reduction Potentials (E0, V) of the [Fe4S4(Sγ-Cys)4]2/[Fe4S4(Sγ-Cys)4]3 (2/3) Quantum Clusters with Substrate and
without Substrate Binding in Wild-Type, No-Tandem and K144A Models
wild-type
2
no-tandem
3
2
K144A
3
2
3
Chain-A without APS
6(FeFe)avg
2.690
2.697
2.711
2.718
2.704
2.698
4(FeS)avga
2.228
2.253
2.229
2.258
2.223
2.253
8(FeS)avgb
2.318
2.334
2.326
2.333
2.329
2.335
4(FeSγ)avg
2.285
2.333
2.299
2.341
2.289
2.329
E
E0
1193.735
1196.724
1185.564
1188.659
1135.356
1138.287
6(FeFe)avg
2.715
2.718
2.728
2.724
2.714
2.706
4(FeS)avga
2.207
2.239
2.217
2.242
2.209
2.242
8(FeS)avgb
2.304
2.308
2.303
2.308
2.306
2.311
4(FeSγ)avg
2.286
2.327
2.289
2.338
2.282
2.319
E
1446.545
1449.460
1438.362
1441.410
1387.923
1390.710
1.35
1.25
1.41
Chain-B with APS
E0
1.43
1.29
1.55
a
The average of the following four FeS distances: Fe1S3, Fe2S4, Fe3S1, and Fe4S2 (see Figure 5). b The average of the following eight FeFe
distances: Fe1S1, Fe1S2, Fe2S1, Fe2S2, Fe3S3, Fe3S4, Fe4S3, and Fe4S4.
the [4Fe-4S] cluster in APSR is much more negative than
ferredoxins, because of the tandem Cys pair and less H-bonding
interactions around the [4Fe-4S] in APSR. There are 10 H-bonding interactions around the [4Fe-4S] active site in ferredoxins.19
These H-bonds are expected to stabilize the negative charges of the
[Fe4S4(Sγ-Cys)4]3 cluster, making the 1e reduction easier. By
contrast, there are only three H-bonding interactions with S or Sγ
in the crystal structure of Pa-APSR (Figure 4).
It is still a big challenge to accurately predict the redox
potentials for the FeS systems. The redox potentials obtained
from quantum mechanical calculations within a solvation model
vary with the dielectric constant of the solvent and the probe
radius for the contact surface between the quantum cluster and
solvent.19,41 In general, the larger the dielectric constant and the
smaller the radius, the reduced state is more stabilized, and the
reduction potential is more positive (or less negative).19,41 Our
previous calculations also show that the DFT/solvation calculations systematically predict the redox potentials of the FeS
systems by 00.5 V more negative than the measured values.19,41
Our current predicted reduction potentials for the [4Fe-4S]
cluster models in Pa-APSR are also very negative, ranging from
1.25 to 1.55 V. Since the experimental reduction potential of
Pa-APSR is not available, we do not have a clear picture if or how
much our calculations overestimate (more negative) these
reduction potentials. Therefore we will focus on the relative
values of the predicted E0’s to see how the APS binding, the
breaking of the Cys tandem pair, and the K144A mutation will
change the reduction potential of Pa-APSR.
From wild-type chain-A to wild-type chain-B, E0 is more
negative by 0.08 V (from 1.35 to 1.43 V). With APS2
nearby, the negatively charged [Fe4S4(Sγ-Cys)4]2 cluster is
then even more resistant to accept an electron from outside, and
therefore is more difficult to reduce.
From wild-type to no-tandem model, E0 becomes more
positive by 0.1 and 0.14 V in chain-A and chain-B, respectively.
Therefore, the no-tandem cluster of [Fe4S4(Sγ-Cys)4]2 is easier
to reduce than the wild-type. This is consistent with our
conclusion in Section 6.2 that the function of the unique tandem
pair Cys139/Cys140 in Pa-APSR is to keep the [4Fe-4S] cluster
more compact and to protect the cluster from reduction.
In chain-A, the calculated E0 of K144A is more negative than
that of the wild-type by 0.06 V. In chain-B with APS, the K144A
model yields the most negative E0 (1.55 V) among all the
model clusters. Upon the Lys144 f Ala144 mutation, there is no
negative charge transfer from the [Fe4S4(Sγ-Cys)4]2 cluster to
Lys144, the cluster is therefore more difficult to reduce, and the
reduction becomes even more difficult when APS2 is present, as
proposed in Section 6.3.
7. CONCLUSION
To understand the role of [4Fe-4S] cluster in APS reduction,
we have examined the coordination and geometry of the native
and APS-bound forms of the enzyme by XAS. Results from the
XANES and EXAFS analysis were valuable in indicating that there
is no change in coordination and overall geometry of the [4Fe-4S]
cluster between both forms of the enzyme. However, in terms of
resolving subtle changes in the geometry and electrostatics, DFT
calculations were employed. Taken together, the EXAFS and DFT
analyses provide a more complete picture of the coordination,
geometry, and electrostatic environment of the [4Fe-4S] cluster in
APSR. This is the first report of the application of both of these
techniques to APSR and thereby contributes to the characterization of the cluster in APSR with a view to gaining insight into the
function of the cluster in APS reduction.
Fe K-edge EXAFS analysis confirms the presence of the [4Fe4S] cluster and a comparison of samples of Mt-APSR in the
native and substrate-bound forms suggests that the core of the
cluster is essentially unaffected during catalysis. This is supported
by biochemical evidence, which shows that the cluster in APSR
has no redox activity during the catalytic cycle,4 and hence we do
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Inorganic Chemistry
not expect any change in the oxidation state of the cluster in MtAPSR with and without APS.
DFT geometry optimizations have been performed on the
[4Fe-4S] clusters of the wild-type, no-tandem, and K144A
models constructed starting from the Pa-APSR X-ray crystal
structures.13 Both substrate-free and substrate-binding forms for
each type of the models were studied. Calculations show that
substrate binding influences the geometric and electronic structures of the [Fe4S4(Sγ-Cys)4]2 cluster, in agreement with the
resonance Raman and EPR spectra experiments.11,18 However,
the geometric changes of the [4Fe-4S] core are not large enough
to be observed in EXAFS and M€ossbauer experiments.
Calculations with the “no-tandem” models show that the
coordinating tandem Cys139-Cys140 pair in Pa-APSR keeps
the [4Fe-4S] cluster more compact and prevents it from
reduction. The tandem pair also leads to a strain in the
geometry of the cysteine side chains. Conformations of the
cysteinyl ligands of an ironsulfur cluster can result in differences in redox energies of ∼100 mV that can directly influence
the redox properties of the cluster.80
Additionally, the replacement of Lys144þ by Ala in our
calculations has a 2-fold effect: (1) Loss of the bridging charged
H-bonding interactions of Lys144 with Sγ-Cys140 and the
terminal sulfate moiety of APS destabilizes the FeS cluster
as the overall charge of the cluster becomes more negative. The
calculated reduction potentials of the clusters in K144A models
are by 0.060.12 V more negative than the wild-type clusters.
In fact, the lability of the cluster and difficulty in reduction have
been observed in EPR experiments in which reduction of the
cluster in the K144A mutant resulted in a 6-fold decrease of signal
intensity and the appearance of an additional signal corresponding
to free Fe3þ formed because of cluster degradation, compared to
wild type APSR.18 (2) Loss of the bridging Lys144 cation increases
the repulsion between the negatively charged APS2 and the
[Fe4S4(Sγ-Cys)4]2 cluster. This explains why the Lys144Ala
mutation is detrimental to APS-binding and catalysis.18
The importance of Lys144 in stabilizing the sulfate moiety of
APS is analogous to the role of a conserved Lys residue in
sulfotransferases which acts as a catalytic acid, stabilizing the
transition state of the substrate, phosphoadenosine phosphosulfate (PAPS), by interacting with the SO3 moiety that is being
transferred.81 Conversely, we could glean a role for the [4Fe-4S]
cluster in positioning Lys144 in the active site such that it can
interact favorably with the incoming substrate. In addition to
Lys144, other conserved positively charged residues such as
Arg242 and Arg245, and Arg171 (present in a flexible “Argloop”) also play crucial roles in substrate binding.9 Lysines and
arginines are cations with long, flexible, and mobile side chains.
They often function as “molecular guidewires” as found in other
sulfate and phosphate transfer enzymes.8287 The cationic side
chains electrostatically screen the bound anions during group
transfer, facilitating covalent bond formation.
It should also be noted that the current Pa-APSR crystal
structure is missing the disordered C-terminal segment of
residues 250267, which carries the catalytically essential
Cys256.13 When the missing segment was modeled into the
active site of Pa-APSR using the structure of 30 -phosphoadenosine 50 -phosphosulfate reductase from Saccharomyces cerevisiae, it
was observed that Cys256 is proximal to the sulfate moiety of APS,
and the side chains of Cys140 and Lys144.18,88 Thus with the
addition of the negatively charged, nucleophilic thiolate in the
transition state, the optimum positioning of Lys144 and other
ARTICLE
cationic side chains to make key H-bonding interactions with APS,
is required to maintain an energetically favorable charge balance
within the active site.
In summary, our study characterizes the [4Fe-4S] cluster in
APSR by EXAFS spectroscopy and by findings from our DFT
calculations which substantiate (1) modulation of the redox
potential of the cluster brought about by the constrained tandem
cysteine coordination, (2) the role of Lys144 as a critical link
between the [4Fe-4S] cluster and APS, and finally, (3) a role for
the FeS cluster in contributing to stabilization of the transition
state via positioning of Lys144 and maintaining charge balance in
the active site during catalysis. To gain further insights into the
mechanism of APS reduction, efforts to determine the structure of
APSR with the intact C-terminal segment are currently underway.
’ ASSOCIATED CONTENT
Supporting Information. Structure-based sequence alignment of Pa-APSR and Mt-APSR, EXAFS analyses of Mt-APSR þ
APS, a comparison of EXAFS of Mt-APSR and ferredoxin and
Cartesian coordinates of the optimized quantum clusters. This
material is available free of charge via the Internet at http://pubs.
acs.org.
bS
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: lou@scripps.edu (L.N.), kcarroll@scripps.edu (K.S.C.).
Phone: (858) 784-2840 (L.N.), (561) 228-2460 (K.S.C.).
’ ACKNOWLEDGMENT
We thank NIH for financial support (GM039914 to L.N. and
GM087638 to K.S.C). The support of computer resources of the
Scripps Research Institute is also gratefully acknowledged. XAS
data were measured at the Stanford Synchrotron Radiation
Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the
U.S. Department of Energy Office of Science by Stanford
University. The SSRL Structural Molecular Biology Program is
supported by the DOE Office of Biological and Environmental
Research, and by the National Institutes of Health, National
Center for Research Resources, Biomedical Technology Program (P41RR001209). We thank Prof. Graham George for
providing the reference 3-Fe and 4-Fe ferredoxin data.
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Supporting Information
A Geometric and Electrostatic Study of the [4Fe-4S]
Cluster of Adenosine-5´-Phosphosulfate Reductase from
Broken Symmetry Density Functional Calculations and
Extended X-ray Absorption Fine Structure Spectroscopy
Devayani P. Bhave,† Wen-Ge Han,‡ Samuel Pazicni,¶ James E. Penner-Hahn,¶‖
Kate S. Carroll,†§* and Louis Noodleman‡*
†
Chemical Biology Graduate Program, ¶Departments of Chemistry and
‖
Biophysics, University of
Michigan, Ann Arbor, Michigan, 48109-2216.
‡
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
§
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458.
Supplementary Figure Legends
Supplementary Fig. 1. Structure-based sequence alignment of APS reductases from Pseudomonas
aeruginosa and Mycobacterium tuberculosis. The ClustalW Multiple Sequence Alignment program
was used. Strictly conserved residues are outlined in red, red letters indicate conserved residues and
conserved regions are boxed in blue. Residues flanking the active site are outlined in green.
Supplementary Fig. 2. Comparison of XAS spectra for Mt-APSR with and without APS bound. A)
XANES spectra; B) k3 weighted EXAFS; and C) Fourier transforms.
Supplementary Fig. 3. Comparison of the k3 weighted EXAFS (top) and the Fourier transforms for
Mt-APSR, [4Fe-4S] and [3Fe-4S] ferredoxins.
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state wild-type 4Fe-4S quantum cluster in
chain-A.
↓
211 atoms
Fe
-0.107163578
Fe
-0.198227030
Fe
2.183164085
Fe
0.718969659
S
1.602736596
S
1.870503940
S
-1.451681704
S
0.537043229
S
-1.211913153
S
4.314827513
S
0.894538389
S
-1.023752245
N
2.814945142
C
2.630658353
C
3.897498754
N
4.760798954
O
4.030531216
C
1.510596542
C
0.141270660
C
-0.833092416
N
-2.148793914
C
-3.202752462
0.141433877
0.084430746
0.143843532
-2.061308632
-1.346877473
-0.965415894
-1.221230441
1.778339840
1.221988848
0.829183148
-4.260319821
0.914816947
-1.131756403
-2.528426347
-3.218330292
-2.437043051
-4.441947797
-2.683361765
-2.201435845
-2.236742036
-1.694652729
-2.359821474
↓
↑
-0.071225829
2.610912383
1.371700639
1.140269589
3.036453877
-0.539619531
1.242881426
1.252125310
4.348255839
1.584941905
0.484547506
-2.007302940
8.352908172
8.028899337
7.493887576
6.824206595
7.651548704
7.002705823
7.471692621
6.296836826
6.606272481
7.066113476
↑
N
N
H
H
H
H
H
H
H
H
H
H
H
H
H
C
H
C
H
C
O
C
-3.144567271
-4.365994470
2.373494881
1.450288698
1.800509062
0.197073979
-0.219560016
-0.939578601
-0.442379369
-2.269069799
-2.377288059
-4.008651983
-4.330207597
-5.072471442
2.558967275
5.977111000
6.869350000
1.997669812
1.743299000
2.707613436
2.599425940
2.788692722
-3.659243998 7.350708690
-1.704665019 7.227461965
-3.061486604 8.949708325
-3.741164636 6.749238435
-2.148361516 6.091952257
-1.173061913 7.844866198
-2.833663836 8.292338539
-3.248711057 5.905642582
-1.617776027 5.489475859
-0.699033027 6.446675358
-4.242500672 7.028988864
-4.144769876 7.567591018
-0.693263267 7.311143786
-2.149914533 7.804807027
-0.423287359 7.666953264
-2.929906000 6.238907000
-2.636805000 6.801957000
5.097724892 0.562735770
6.022431000 0.036064000
5.349097582 1.874270553
4.582996234 2.842153837
4.158455198 -0.366632709
C
N
C
N
C
H
H
H
H
H
H
H
N
C
H
C
H
C
N
O
C
H
H
H
C
C
4.171407136
4.493900142
5.813090571
6.349424273
5.364859830
1.056352157
2.206420649
2.861429080
3.835502010
5.578245489
6.343711652
7.349099906
1.851781405
1.539951000
1.476316000
0.602304989
0.982050000
1.756295478
2.579895950
1.946715695
-0.445495449
0.130933071
-1.267965945
-0.025709614
3.755613999
4.649552585
4.562525602
5.599935530
5.684451499
4.723192536
4.013406729
4.596539551
4.034656240
3.180403701
6.213091144
3.169285533
6.396304492
4.554952497
2.441429385
3.763437000
4.465196000
3.062243297
4.110914000
2.068372025
2.117495110
1.271931071
2.903266299
2.918322961
3.595272964
3.180271091
1.264894101
1.385011288
-0.726089756
-1.579299724
-1.732589854
-0.992455757
-0.357905298
0.795646930
-1.283992115
0.105529851
-2.053261167
0.274847642
-2.343190209
-0.911127970
-6.986286499
-6.462627000
-7.300786000
5.631491151
5.606559000
5.591746219
4.517683197
6.524545166
4.522090288
6.608355728
4.738612644
3.555072790
4.414402282
5.642420468
N
O
H
C
H
H
H
H
H
C
C
N
O
C
H
H
H
H
N
C
C
N
O
C
H
H
H
C
H
C
C
H
N
O
C
O
C
H
H
H
H
H
H
H
C
H
C
O
C
4.852940320
5.249836736
2.439928186
4.640716655
3.455742591
4.632799117
5.668849935
5.323203000
0.920073657
-0.066652883
-1.121586470
-2.216216683
-1.020850860
-0.314108093
-0.133716460
-0.269354869
-1.318440501
-3.034079000
-2.438732123
-1.379217093
-0.652499495
-0.973879323
0.198477658
-0.356135303
-1.830818095
-0.031269820
0.514728034
-0.358249000
-1.111311000
1.008812071
-0.384875398
1.466691000
-0.963179494
-0.942734331
1.131519288
0.177215033
2.556222095
1.596924646
0.908070043
0.282656739
2.742078412
2.726874286
3.272466642
-1.938787000
3.080778000
3.027971000
2.900252454
3.679943971
4.468020634
2.632560987
0.409927522
2.834285655
1.629976360
0.217932421
2.716748419
1.347484651
2.690085000
-5.162294918
-5.299498191
-5.053704819
-5.823288715
-4.126746215
-4.241222707
-6.310482364
-3.259866189
-4.369094126
-5.540736000
-1.134991812
-0.256284965
-0.722114230
-1.936988002
0.015071503
-0.077456745
0.706940452
-1.059717558
0.422897408
-2.570636000
-2.639926000
-6.173433679
-6.389709039
-7.130947000
-7.540668443
-5.591419167
-5.429452514
-5.994180754
-5.597507510
-5.666257177
-4.366180708
-5.482242524
-4.976101329
-6.647691473
-5.320028509
-7.684524000
0.711402000
0.637566000
2.169919822
3.049098994
0.178325561
6.084277666
6.109672809
3.810551462
3.206528167
4.357267917
3.075059041
3.455062010
6.970951000
-2.422680393
-1.970367177
-3.021283633
-2.950627555
-3.844024174
-0.890724195
-1.556226841
-1.363657588
-0.472819566
-3.483255000
-4.093678569
-4.523455290
-5.791526197
-6.245323764
-6.305377188
-3.401063682
-4.775303509
-3.047128378
-3.822098570
-7.389921000
-8.181831000
5.268075699
5.799845004
5.085305000
5.424603407
6.571487584
3.942096624
3.030418686
3.434136432
6.033382962
4.084971676
2.184997661
2.557680914
3.164624520
4.209591446
5.661721000
-8.141230000
-9.232461000
-7.776099284
-8.193160390
-7.745682969
C
C
N
C
N
N
H
H
H
H
H
H
H
H
H
H
H
H
N
H
C
C
C
C
N
C
C
C
N
C
C
C
C
C
C
C
H
O
C
O
H
C
O
H
N
C
N
N
C
4.663564514
4.070244442
4.758434579
5.882274403
6.492114473
6.386241751
2.282915943
4.658892271
5.216469753
5.735459257
4.213587556
4.085046523
3.024185533
4.419245054
6.474154410
7.393642776
5.823963652
7.227176185
3.516609831
4.046672000
6.235497073
5.334747867
5.589419338
6.883712395
7.032691457
2.580952725
2.954932322
2.082111902
2.743904218
4.068334810
4.243531899
5.121997219
6.386890755
6.594239144
5.541712394
0.165471568
0.736896000
1.053932726
-3.748830460
-4.265410215
-4.321424000
3.223803242
3.526790657
3.237939000
5.756789645
6.867234570
6.950339478
7.891919616
9.146220609
-1.305307100
-2.278220530
-2.248011769
-2.888661125
-2.576179494
-3.836250940
0.102886085
0.366107375
0.752373463
-1.519883670
-1.531705317
-3.301016899
-2.059369455
-1.586246986
-1.595008641
-3.016624988
-4.284156895
-4.322930812
6.419167490
6.552932000
-2.572263417
-3.314926617
-3.016355742
-3.610234684
-3.320651862
4.279902513
3.272894846
2.636321616
1.693858144
1.701915111
2.696525269
0.935099373
1.173154207
2.163542071
2.924976485
-3.973511496
-4.386578000
-3.969267659
-0.834269085
0.171240276
-1.617048000
-0.686823622
-1.389571173
0.408880000
4.764352652
4.052777097
3.384894639
4.092253793
3.357720176
-8.075127872
-7.058709543
-5.765085018
-5.446714106
-4.282724603
-6.222750176
-7.716137729
-6.681676941
-8.301051296
-8.169724586
-9.051496357
-7.445243215
-6.840047832
-5.076180558
-4.022695565
-4.118710936
-6.936499629
-5.931659799
1.929881111
2.783092000
4.770871983
3.791081710
2.311386526
1.791757732
0.328656602
-5.443270928
-4.397709648
-3.551154712
-2.808200805
-3.140667291
-4.141007179
-2.646523870
-3.161085637
-4.139600584
-4.622321091
-7.039407791
-7.879897000
-5.919961232
-4.106473549
-4.602123323
-3.604721000
9.550738005
10.519820306
9.561120000
-9.458928009
-9.214152532
-8.055136326
-10.053817752
-9.894941087
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9.027970000
8.816378000
4.606322935
1.289349613
4.222338319
-2.334886945
-1.742686214
-0.552603981
3.588649907
7.286380968
6.145531238
4.294107104
5.434648259
5.581927526
4.758706422
6.906682387
7.770410690
7.903395296
7.064663363
6.255233343
0.552805203
2.161014731
3.477162461
1.020737636
2.325521041
4.956290849
7.236971459
7.601996806
5.729518326
-2.172488533
0.452021815
-0.684370702
0.482359550
9.986308614
8.255758348
9.848710157
9.537807830
7.742536692
5.640395562
4.897827947
7.715228938
6.082748757
5.940755698
1.861262000
1.617659000
-1.433329427
1.661825885
3.385838804
-6.515185150
-2.422868962
-8.129194086
7.104680572
-2.832625758
-1.490834549
-3.055639933
-4.394895724
-1.932679896
-3.430807948
-4.699439297
-3.186817548
-3.720121307
-2.308289355
-3.724220311
3.692281051
5.188438580
4.586258403
2.779102712
1.109659627
0.202740719
0.608694237
2.339048499
3.711167585
-1.987523830
-1.925039689
-4.643658364
-3.930208400
1.393993448
1.392566721
3.815331744
3.551511106
4.591016409
5.139266418
4.424175581
2.743411105
3.172945084
-4.022585404
-10.151452000
-11.197833000
6.779211379
-6.657338257
5.838075662
-2.219478947
-5.796025079
4.709579256
1.188648967
4.588721753
4.628996021
3.998732344
3.959400056
2.146261581
1.725904620
1.885797451
2.266908576
-0.044139584
0.158980088
-0.208841311
-5.999322601
-4.988395253
-5.987057630
-3.415421949
-2.092982607
-1.864025746
-2.783728876
-4.508360745
-5.345487333
-3.605853601
-7.728135736
-6.849853435
-5.123834966
-9.893014236
-9.535621969
-10.596431199
-8.889413542
-10.926339732
-10.395140818
-9.028949725
-7.897039453
-7.578382102
6.323985157
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state wild-type 4Fe-4S quantum cluster in
chain-B.
↓
↓
↑
↑
250 atoms
Fe
19.396986524
Fe
17.491248673
Fe
19.860088525
Fe
17.947835850
S
17.748790882
S
20.186105800
S
17.106275331
S
19.640578559
S
15.705038845
C
15.187628311
S
20.546738678
C
19.950291474
S
21.545425264
C
21.016767824
S
17.185530524
C
18.563313865
H
15.401924271
H
14.097840315
H
20.813394426
H
19.200518618
H
20.543256168
H
20.255748471
H
18.936826763
H
19.390184996
C
24.644002000
C
24.845930179
N
26.269407614
C
26.794123151
N
28.117585890
N
26.020689515
H
25.141612000
C
21.415000000
C
20.194111459
O
20.121338576
C
22.650295227
C
23.944641029
C
24.030438443
N
23.916835743
C
24.880950323
N
24.552019493
N
26.113629246
H
21.618741000
N
19.217146883
C
17.991000000
C
18.151813509
C
18.922565079
C
18.614773893
67.098967432
65.134308275
64.718218139
66.361463918
64.089553997
66.821240370
67.301134090
65.036720377
64.723061435
66.287429310
68.741982740
68.964543351
63.383797719
63.084584721
67.155427901
67.178537028
67.112450703
66.226040468
69.331817107
69.763069450
62.098157312
63.823295137
68.197583141
66.555090194
76.620997000
75.376409984
75.163829772
74.277507187
74.089232722
73.572117499
76.554791000
71.340996000
72.135296907
73.363447142
71.697081896
71.070145353
69.548299219
69.056605444
69.126685637
68.822580444
69.473695689
71.609153000
71.415462385
71.973996000
72.662023593
71.854839761
70.589553441
56.308529360
56.458016892
55.162330743
54.119847136
54.485618459
54.263555910
55.914209267
57.306009251
57.835590561
58.662006741
57.442095716
59.169319863
54.373596091
52.654975810
52.149731675
50.949742155
57.983973318
58.774929012
59.732089410
59.186622107
52.610310241
52.400140163
50.863881769
51.298879912
48.376996000
49.210413132
49.449448202
50.290095587
50.311037580
51.119631307
47.403845000
47.975999000
48.397864541
48.214689961
48.805287323
48.285561440
48.385920270
49.758566157
50.681964258
51.960672980
50.372639712
46.934074000
48.977583760
49.510998000
50.876927515
51.864350043
52.288524132
C
N
C
C
C
C
C
H
C
C
O
C
C
N
C
C
N
H
N
H
N
C
C
O
C
C
C
N
C
N
N
C
O
H
N
C
C
C
C
C
N
H
C
C
O
C
O
C
H
20.177763167
19.607035928
20.579729877
21.001100836
21.764522518
22.175539535
22.556326836
17.633684000
15.701382029
16.053536848
16.252781943
16.950896225
17.875906551
17.653274222
19.070772952
18.660865591
19.524933619
15.078911000
16.167657421
16.562673000
19.519478233
20.351869421
21.806877271
22.696658769
19.759116140
18.382648588
17.844573193
16.534778938
16.288781970
15.017292526
17.266109416
19.374936036
19.937676049
18.677061000
22.005157589
23.289001000
23.264095182
24.672364252
24.690562726
24.510292807
24.035258723
23.715284000
22.360184129
21.387038063
20.249697795
22.129997260
22.213666694
23.184562672
23.378128000
72.187068369
70.115101389
71.065293386
73.321651051
71.049843176
73.311847512
72.185370673
72.744707000
70.892953061
70.966667924
69.942471805
70.820873427
71.975016768
73.209609703
72.086060419
74.036265692
73.361415676
71.721456000
72.194485066
72.275889000
63.813261328
62.825875435
63.280168505
62.420223558
62.454787028
61.796282376
61.691352764
61.058375909
59.753229927
59.337941490
58.854707009
63.925411899
63.236059925
64.732842000
64.599352565
65.171998000
66.029566290
66.293794464
67.205985668
68.671315453
69.471110984
65.719872000
60.395788286
59.245191518
59.305704842
61.509961392
60.895501450
62.594564831
60.073913000
52.464621182
53.106398602
53.237449935
52.447823218
53.967609294
53.182129798
53.932151753
48.820090000
56.288805692
57.766042366
58.434933890
55.391592375
55.496220045
54.921005235
56.143637874
55.187964230
55.935571129
55.939197000
58.307868137
59.244499000
62.721583918
62.071257347
61.855563201
61.770966662
60.711330601
60.756924706
59.330999065
59.226958123
59.164526764
59.009201601
59.229946030
64.050326600
64.908847909
64.291576000
61.692091827
61.339999000
60.079461871
59.546393651
58.314685427
58.682927025
57.506494364
62.186842000
58.494271392
58.382256246
58.877241851
57.482898780
56.185404259
57.659369117
58.312270000
N
H
C
C
H
O
N
C
C
O
N
H
N
C
C
O
N
C
C
C
O
H
N
H
S
O
O
O
P
O
O
O
O
C
C
O
C
O
C
O
C
N
C
N
C
C
N
N
C
21.825670346
21.170100000
15.761842550
17.268419745
15.420306000
17.829993240
17.959628369
19.391255849
19.778271831
20.854905477
18.970037379
19.238078000
17.906977406
18.116917951
19.123272949
19.308887271
19.753945808
20.853000000
22.140884764
21.419697368
20.972542621
22.652465000
21.165454838
20.434047000
26.544787925
26.726606507
25.901168095
25.983430166
28.547583783
28.023532330
28.284174150
28.136794439
30.170053174
30.911761898
31.969981316
31.406734743
33.140375064
34.283600077
32.649455837
33.726284211
31.857351143
30.703432600
29.383293175
28.621811305
29.499822196
29.332066736
28.130329118
30.418666028
31.608709721
58.151095947
57.394186000
66.580745229
66.551507956
67.594068000
65.679613919
67.587740468
67.729973609
67.856211768
67.404520461
68.576270784
68.583743000
65.286498694
66.714469250
67.223614643
68.443360538
66.295335142
66.607000000
63.162679876
63.005104960
63.983919272
64.135944000
61.746403988
61.601916000
70.950791719
71.545774616
71.823329495
69.581027396
70.758736253
69.493652753
72.106810480
70.768161188
70.711621912
69.485352408
69.725471261
69.626582660
68.758910110
69.419688374
67.651938499
66.892364179
68.444452601
67.665164372
68.028763525
67.097255057
66.070766826
64.787718190
64.297296079
64.000620802
64.456627933
57.739198965
57.563113000
60.053453633
60.236933389
60.325163000
60.912921138
59.698131676
59.900144499
61.375452974
61.783494877
62.161668400
63.137216000
49.557053890
49.563900818
48.526606310
48.417378403
47.801817296
46.924000000
51.610917956
50.294948831
49.672924569
51.642252000
49.902273878
49.210267000
55.146700609
56.479295352
54.164334480
55.191159816
53.004917751
52.371179532
52.394809915
54.635858380
53.201117709
53.369332931
54.432471172
55.778516284
54.415007444
54.966319742
55.334617488
55.845494444
56.393549908
56.891911440
56.992291950
57.533295250
57.811560899
58.365773086
58.715226309
58.512573737
58.101803761
N
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
31.889922395
30.794797136
15.356828236
18.951314708
17.461095080
19.899958956
17.165024398
19.530107036
22.874246697
21.426521526
23.008073127
23.163693224
24.181738242
21.968711641
21.130365235
22.306822217
22.717001954
17.035761064
18.240873223
14.832225627
14.279828693
15.739731549
18.551354180
17.714592346
18.458949104
17.678800989
19.715911835
20.456346620
20.407025551
21.236772458
23.956355311
22.685461103
22.743903890
25.308663239
25.121090787
25.638868012
23.889670693
23.757982330
25.446889576
24.021247063
24.659661568
23.646187853
25.282431591
26.839424909
26.425818208
23.130777797
23.220953037
24.961298260
24.085129434
65.638118864
66.406751493
65.875885599
64.429253705
68.302341531
66.831975741
67.186999427
65.320398972
62.365506911
60.954370555
63.424926555
62.984817961
62.181183398
61.601946812
61.938668466
60.789883283
58.139265253
57.870843876
59.103091744
58.351101358
59.955168342
61.676884093
61.170457986
62.694138844
60.804248182
62.387721488
63.361171199
61.772066618
61.943768765
65.249154683
64.320537949
65.508508706
66.964547820
66.721027277
65.333138877
67.091732914
66.891762897
68.820104042
69.125360991
70.471594862
69.366958544
69.159519311
68.937147847
69.548712962
69.409607658
68.452301149
69.072250304
69.190020495
71.327487974
57.544144527
57.427613956
60.784356066
62.142119536
59.175310440
59.551252763
49.303297611
47.970100477
51.771045730
50.478640230
56.972053224
58.679482855
57.461473545
55.533693151
57.617904305
59.511038640
57.257543586
59.140913759
59.082678620
59.164523184
59.340808228
59.097041544
58.682476010
58.925173680
61.217909919
61.352947257
60.098389412
60.227135789
62.716598153
61.826712866
61.179718639
59.309435021
60.289004881
60.334274488
59.267159221
57.776024049
57.631682888
59.458990847
59.017715763
57.742511149
56.681648254
52.272251711
52.660121501
51.111071418
49.411215452
49.972623549
47.833200861
47.931076124
47.226257964
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
O
H
O
C
H
H
24.789247107
22.765427115
22.487689171
21.203761866
19.341735684
17.254408119
18.656112687
17.140616146
20.725284767
22.818993571
23.494806359
22.044474977
19.617667752
17.756918822
20.400753710
18.756981064
16.855589088
19.631872289
17.515528934
16.608711952
15.131598887
16.080821875
33.311004617
34.796255456
28.093392159
27.347105479
32.449592476
29.040975660
30.251577101
31.399138217
26.484340464
25.090212443
28.472525127
28.657717887
26.918920039
24.335856646
24.427581901
23.571378959
25.021611851
32.479176181
32.341042763
18.007694338
16.838216594
15.946914982
16.849535000
22.483155038
22.147720413
22.982507000
18.555141174
71.507344509
72.785086781
71.426479221
70.270259632
70.411478332
71.167319764
73.619726567
72.893416620
74.198549159
74.188850014
72.199264229
70.182473203
69.197341719
69.971582197
73.741304636
75.056426819
73.464094381
71.355360378
69.923627306
70.719047961
69.971496378
73.038109146
66.283923190
68.720389754
63.445710540
64.930556348
63.781509095
68.987677694
68.657959605
69.260421163
72.976251121
73.904096154
73.349085849
74.318591572
75.644053045
75.506877489
74.502531778
76.760972567
77.502562817
68.684317196
70.751559692
68.774544391
64.670137276
65.209684662
63.603479000
66.155241398
65.901909795
66.274602000
64.704994599
48.829815815
48.780704504
49.855951939
48.011784871
49.043383110
49.560648175
50.722171629
51.241536479
51.869187242
53.183178092
54.470440676
54.555351173
53.534022542
52.074894962
56.285022328
54.855962395
54.344319946
56.702566178
55.635131238
54.356331631
56.140459758
57.754236138
56.517684310
55.422860755
59.264289741
58.775030051
58.239793493
56.637158106
53.639567481
52.412622769
51.807877723
51.341727364
50.919988038
49.486804593
48.834413259
50.171118487
48.700510842
48.197883528
48.906516013
57.265515186
54.328395847
61.916544553
49.021342256
48.360524830
49.253523000
48.716781014
47.348931113
46.742711000
50.082679478
H
H
H
H
H
H
H
21.978260652 65.485264567 49.231225244
20.601667000 66.337714000 45.892815000
20.989612330 67.688249151 46.959665998
22.059246237 64.822055920 47.173568752
33.363738234 68.382795240 53.410059769
31.945659303 67.018323545 54.774408759
23.061204346 69.198900309 57.272926054
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state no-tandem 4Fe-4S quantum cluster
model in chain-A.
↓
211 atoms
Fe
-0.054305588
Fe
-0.233227045
Fe
2.252523730
Fe
0.746584959
S
1.598885362
S
1.938094773
S
-1.433257776
S
0.567148204
S
-1.360304293
S
4.441290543
S
0.878305799
S
-0.975799392
N
2.800264022
C
2.605261795
C
3.864941677
N
4.760765971
O
3.957259539
C
1.473685136
C
0.103447518
C
-0.883608505
N
-2.196354629
C
-3.251783510
N
-3.193075815
N
-4.413754961
H
2.352590945
H
1.411829735
H
1.758572637
H
0.147418516
H
-0.243108255
H
-0.990344754
H
-0.503861636
H
-2.324976845
H
-2.434794512
0.187557929
0.069135846
0.164000675
-2.039967749
-1.324948891
-0.929528652
-1.210288992
1.784602845
1.063956262
0.775711068
-4.242168134
0.960741198
-1.131090654
-2.523300186
-3.214278324
-2.426110808
-4.445559496
-2.665891924
-2.191291774
-2.244268925
-1.697693831
-2.358867188
-3.655219219
-1.701918608
-3.059877539
-3.721651484
-2.130534481
-1.161416286
-2.822495497
-3.262435788
-1.637267751
-0.710974308
-4.244023119
↓
↑
-0.074383955
2.597468994
1.412340746
1.144084076
3.062351120
-0.515173723
1.183730111
1.318654212
4.341706409
1.577998432
0.499432685
-2.020526024
8.352587838
8.017308721
7.462973284
6.838356325
7.556356601
7.000232026
7.478614305
6.312794986
6.625129800
7.088988779
7.389087098
7.243152894
8.937229149
6.739634140
6.086746094
7.852276123
8.306009588
5.937776339
5.491269253
6.418538100
7.056186479
↑
H
H
H
H
C
H
C
H
C
O
C
C
N
C
N
C
H
H
H
H
H
H
H
N
C
H
C
H
N
C
H
H
H
C
C
N
O
H
C
H
H
H
H
C
C
H
N
O
C
-4.059262356
-4.382023111
-5.127747885
2.493616736
5.977111000
6.869350000
1.983263985
1.743299000
2.665096262
2.484141005
2.781562359
4.155481572
4.456991754
5.773797879
6.328585657
5.358403055
1.036450325
2.196993128
2.870340739
3.785488240
5.587184232
6.290230564
7.330851456
1.856379490
1.539952000
1.476316000
-0.169694739
0.090269947
2.879477152
-0.842322451
-0.828418444
-1.753725390
-0.176058591
4.123943506
4.959892501
4.738762462
5.792384059
2.831354014
4.999731091
3.915577662
5.092025273
6.005014614
5.186885107
-0.092483957
-1.131084440
0.901281896
-2.233625343
-1.012035305
-0.326010410
-4.135323602
-0.689679241
-2.144382235
-0.423185129
-2.929905000
-2.636804000
5.099406029
6.022431000
5.362945301
4.641167620
4.160311977
4.581335179
5.632523761
5.735452727
4.771951407
4.041815753
4.603373820
4.021123565
3.186970221
6.241237752
3.192464417
6.463240845
4.616438419
2.444910916
3.763439000
4.465198000
3.103937559
4.171509837
1.977430652
2.827025200
2.871841652
3.430185630
3.105140015
1.224796361
1.514656233
2.715986493
0.697969768
2.664971600
1.496334487
0.152792081
2.582435122
1.099014206
2.962073568
-5.307275203
-5.067184910
-5.201978921
-5.824998279
-4.154914810
-4.219279522
7.610317374
7.314527410
7.813132591
7.693025133
6.238906000
6.801956000
0.567105016
0.036064000
1.893812130
2.882507578
-0.355094907
-0.726722725
-1.570674723
-1.736388270
-1.012229410
-0.377543282
0.787541904
-1.269198809
0.123525515
-2.031955702
0.242772236
-2.340692498
-0.934006958
-6.989848484
-6.462626000
-7.300785000
5.941767704
5.993627562
4.429586025
4.604188971
6.785436477
4.511967292
3.787277472
4.414396097
5.676942832
6.230416966
6.102233149
3.679491950
3.183248747
4.451852178
3.057340726
3.359471668
7.108268522
-1.930785919
-2.997561423
-2.376172110
-2.930413994
-3.834274992
-0.878781451
H
H
H
H
N
C
C
N
O
C
H
H
H
C
H
C
C
H
N
O
C
O
C
H
H
H
H
H
H
H
C
H
C
O
C
C
C
N
C
N
N
H
H
H
H
H
H
H
H
-0.184907014
-0.258713340
-1.335104447
-3.034078000
-2.442820433
-1.387261474
-0.670922629
-0.975702459
0.160289872
-0.350215916
-1.841924425
-0.034948465
0.522381101
-0.358249000
-1.111310000
1.001816669
-0.390449592
1.466690000
-0.966124065
-0.950638867
1.111689642
0.139375143
2.527117964
1.592709080
0.896947096
0.238152666
2.707884080
2.681063738
3.256247365
-1.938788000
3.080778000
3.027971000
2.910753663
3.703910707
4.463336007
4.667614770
4.080541286
4.766138077
5.892582456
6.516861987
6.382229487
2.278288409
4.646909220
5.215519080
5.741321934
4.222330816
4.107625516
3.031697137
4.435961278
-6.306000568
-3.249554942
-4.317166675
-5.540736000
-1.116899780
-0.239873411
-0.710182532
-1.935081649
0.036638998
-0.058854586
0.722133377
-1.042094907
0.425052359
-2.570636000
-2.639926000
-6.171714610
-6.385474176
-7.130947000
-7.538634200
-5.580500281
-5.429915583
-5.984605754
-5.609862914
-5.659187648
-4.365276385
-5.469377808
-4.988002190
-6.661056010
-5.343459583
-7.684524000
0.711404000
0.637569000
2.171416163
3.046463313
0.172338378
-1.306865921
-2.297048351
-2.277117946
-2.916874443
-2.588197876
-3.880183800
0.107688748
0.350457159
0.751889046
-1.513205031
-1.525550372
-3.313775867
-2.091276479
-1.609396997
-1.492576735
-1.372668268
-0.463725511
-3.483256000
-4.084999374
-4.527390682
-5.802881582
-6.242808980
-6.334700589
-3.415463146
-4.779965034
-3.055348923
-3.851816115
-7.389921000
-8.181831000
5.264340215
5.799349139
5.085306000
5.428219567
6.562494761
3.935748650
3.036609199
3.408145408
6.023774534
4.082443178
2.192563083
2.531365550
3.133105567
4.175067202
5.661722000
-8.141230000
-9.232461000
-7.770561531
-8.173942581
-7.742061871
-8.086138982
-7.082890464
-5.787745602
-5.473571011
-4.323185835
-6.239277799
-7.717228601
-6.675032616
-8.286070327
-8.179448589
-9.066381426
-7.483359521
-6.864166393
-5.100100131
H
H
H
H
N
H
C
C
C
C
N
C
C
C
N
C
C
C
C
C
C
C
H
O
C
O
H
C
O
H
N
C
N
N
C
C
H
H
H
H
H
H
H
H
H
H
H
H
H
6.506485156
7.410957834
5.808798227
7.235088097
3.506989762
4.046672000
6.213249472
5.312443095
5.569018020
6.874198354
7.039142806
2.575211531
2.964231093
2.102590481
2.776333914
4.097644078
4.258270387
5.159701851
6.418000523
6.610305499
5.549543361
0.156488114
0.736896000
1.030898201
-3.755649323
-4.277123230
-4.321424000
3.217636837
3.546011118
3.237939000
5.710472249
6.798316424
6.813878952
7.857025494
9.109012949
9.027971000
8.816379000
4.659694433
1.285725365
3.897611597
-2.376460773
-1.735717362
-0.554065312
3.661730968
7.265667518
6.110801146
4.270874043
5.414047568
5.548222310
-1.604552792
-3.040174926
-4.341745508
-4.353364278
6.412083899
6.552931000
-2.566414694
-3.323447256
-3.045755981
-3.631393488
-3.346703534
4.284372928
3.273883379
2.628879446
1.687638206
1.704098238
2.703438266
0.940451204
1.187062228
2.180862560
2.938106589
-3.975671414
-4.386578000
-3.981667103
-0.825623575
0.176697755
-1.617049000
-0.684748171
-1.388947740
0.408881000
4.793739343
4.065509779
3.399646174
4.086939684
3.360773319
1.861263000
1.617661000
-1.417762731
1.665828090
3.209829694
-6.496188550
-2.427358766
-8.128515625
7.024748032
-2.811486471
-1.485115746
-3.064078886
-4.400556877
-1.965355165
-4.073254015
-4.151842549
-6.935070565
-5.960898886
1.935009596
2.783093000
4.772534741
3.803558074
2.320845505
1.819833899
0.357322321
-5.441427451
-4.405191006
-3.553969355
-2.820537996
-3.165089167
-4.163454790
-2.684313814
-3.210858547
-4.189036079
-4.660031642
-7.041520453
-7.879897000
-5.911460272
-4.102669684
-4.598652146
-3.604721000
9.552516474
10.508668972
9.561120000
-9.601616052
-9.309430110
-8.145294241
-10.106308754
-9.889356273
-10.151451000
-11.197832000
6.900014619
-6.674772885
5.944268955
-2.184397395
-5.785691864
4.715323569
1.144876878
4.579080900
4.637200150
4.006965574
3.988030482
2.137863756
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4.746779806
6.906484726
7.751541081
7.925166101
7.049456479
6.281037058
0.550921920
2.143952725
3.466774344
1.041786695
2.366531188
5.005112216
7.274351047
7.613240190
5.725721241
-2.173323516
0.451754322
-0.698267513
0.451554033
9.998577632
8.268654482
9.835978643
9.455173570
7.768548159
5.619723858
4.837998545
7.565385544
5.908642553
5.927193536
0.757080428
2.091114258
-3.479796176
-4.719847418
-3.198450684
-3.726670093
-2.334834731
-3.770537822
3.688359502
5.183054892
4.607474650
2.764565926
1.095026669
0.203402874
0.624830526
2.361582755
3.720188463
-1.964064780
-1.925026171
-4.643861903
-3.958306640
1.419762272
1.371700425
3.826751686
3.558951340
4.627674356
5.152148882
4.502964206
2.751708154
3.166250331
-4.020124188
2.534928354
1.351279616
1.737786036
1.920285881
2.303599634
-0.000191396
0.181357525
-0.192134324
-6.002791220
-4.977848634
-5.983133155
-3.407352935
-2.107216664
-1.903974239
-2.844609610
-4.568373773
-5.390602650
-3.591123245
-7.726906889
-6.867091615
-5.119466007
-9.897226239
-9.534406515
-10.559834336
-8.867987605
-10.962250495
-10.546743854
-9.164508343
-7.953068903
-7.748524232
6.325910432
6.043702636
4.305134025
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state no-tandem 4Fe-4S quantum cluster
model in chain-B.
↓
250 atoms
Fe
19.456112321
Fe
17.468860469
Fe
19.901278452
Fe
18.041597871
S
17.832380510
S
20.280209298
S
17.185539923
S
19.563126340
S
15.669434913
67.170510792
65.170955277
64.776935263
66.417142105
64.137391636
66.880441540
67.373512771
65.092297317
64.523085737
↓
↑
56.435488094
56.453879129
55.267920603
54.187823400
54.497964333
54.396663481
55.973542990
57.428882544
57.696080082
↑
C
S
C
S
C
S
C
H
H
H
H
H
H
H
H
C
C
N
C
N
N
H
C
C
O
C
C
C
N
C
N
N
H
N
C
C
C
C
C
N
C
C
C
C
C
H
C
C
O
14.835102764
20.637231300
20.250436524
21.535699484
20.997922644
17.249937591
18.587456729
15.227685202
13.777163766
21.200302947
19.627147836
20.511607934
20.247385072
18.973037052
19.416044698
24.644002000
24.830976692
26.253059585
26.769430877
28.086889616
25.984422689
25.141612000
21.415000000
20.197436723
20.127545323
22.651803670
23.937421076
23.997283298
23.881224712
24.852334917
24.525425630
26.090556857
21.618741000
19.219848889
17.991000000
18.144828081
18.913574235
18.600512122
20.173472544
19.594624635
20.572358690
21.002905597
21.759217479
22.179840443
22.558401295
17.633684000
15.680715526
16.011006914
16.122887853
65.899928761
68.830715680
69.064739264
63.421780399
63.125223015
67.131883171
67.159436798
66.849105855
65.844245299
69.275315237
69.963948759
62.144058098
63.873524158
68.175663917
66.516878290
76.620996000
75.368758120
75.145968850
74.226759700
74.014741892
73.504573794
76.554789000
71.340996000
72.138310005
73.367684123
71.684202558
71.032259295
69.511342881
69.049501357
69.105841972
68.823866651
69.417034819
71.609153000
71.417870003
71.973996000
72.655003789
71.840508854
70.573108725
72.159414687
70.084770066
71.028194911
73.289923324
70.998398579
73.266318869
72.129614896
72.744706000
70.883580470
70.961807766
69.938520940
58.606509228
57.551842384
59.344382839
54.400667331
52.679741971
52.198271664
50.946768512
58.236299600
58.323611989
59.845345863
59.437102114
52.643037734
52.424014663
50.866733990
51.257608774
48.376995000
49.200860005
49.436612080
50.246813202
50.252915487
51.055271542
47.403844000
47.975999000
48.400217860
48.220180856
48.807855657
48.297336416
48.425813852
49.807753478
50.723577372
52.008227691
50.401997061
46.934074000
48.978510811
49.510998000
50.880881235
51.863403306
52.276941519
52.461036344
53.084246569
53.221605886
52.453557621
53.948078390
53.183712833
53.920267100
48.820090000
56.291553002
57.773698082
58.460325539
C
C
N
C
C
N
H
N
H
N
C
C
O
C
C
C
N
C
N
N
C
O
H
N
C
C
C
C
C
N
H
C
C
O
C
O
C
H
N
H
C
H
N
C
C
O
N
H
N
16.922812190
17.867745637
17.662168484
19.071227405
18.688701733
19.547010980
15.078911000
16.187320837
16.562673000
19.510963601
20.335021542
21.789038131
22.657822854
19.730649297
18.369065780
17.853040544
16.553572645
16.321841927
15.054973608
17.308052040
19.387833385
19.993002141
18.677061000
22.003092543
23.289001000
23.253766529
24.662386901
24.692253983
24.557158354
24.056991213
23.715284000
22.357872828
21.387531451
20.246800815
22.129794648
22.205572742
23.189493988
23.378129000
21.829276909
21.170101000
14.967872766
14.342282492
18.126729919
19.546015423
19.779441266
20.895900943
18.699626597
18.739084701
17.877069479
70.791275159
71.928103608
73.165533081
72.014960155
73.972148749
73.281252995
71.721456000
72.193249810
72.275889000
63.809786437
62.824880605
63.275193136
62.408756452
62.461540855
61.771678861
61.623383493
60.971898739
59.662388560
59.239838428
58.773934746
63.936287005
63.275100933
64.732841000
64.599790284
65.171998000
66.027649626
66.258762308
67.158561528
68.629494646
69.438544304
65.719872000
60.395068119
59.243686462
59.306856939
61.505057089
60.889861080
62.585112485
60.073913000
58.146914452
57.394186000
65.827169920
66.610732638
67.799583738
67.904579323
68.040816533
67.827795796
68.411072054
68.468973379
65.299951869
55.388877122
55.484096195
54.909420323
56.118597449
55.163555699
55.903165035
55.939197000
58.298261584
59.244499000
62.711777051
62.045714884
61.811248669
61.648451451
60.688383016
60.727794938
59.295989123
59.191611262
59.130198975
58.982386565
59.194461661
64.047902263
64.893506487
64.291576000
61.705077013
61.339999000
60.080806267
59.529034068
58.288835681
58.651069370
57.493566386
62.186842000
58.482014704
58.369914389
58.857804701
57.465500368
56.168169228
57.638472904
58.312270000
57.736100646
57.563113000
60.122778369
60.575214208
59.745536044
60.056926733
61.577582677
62.075882272
62.278971658
63.291823715
49.540549861
C
C
O
N
C
C
C
O
H
N
H
S
O
O
O
P
O
O
O
O
C
C
O
C
O
C
O
C
N
C
N
C
C
N
N
C
N
C
H
H
H
H
H
H
H
H
H
H
H
18.099288330
19.102047874
19.278157625
19.750132907
20.853000000
22.118341495
21.387619647
20.916440085
22.652465000
21.160900645
20.434046000
26.573594656
26.872958723
25.963577534
25.888174877
28.496817793
27.989254872
28.189844362
28.113610466
30.120181113
30.873890045
31.917000008
31.350126332
33.099627987
34.225225188
32.615389095
33.696063759
31.813249769
30.665695764
29.342858298
28.584971559
29.468702394
29.304798581
28.099659040
30.395316119
31.584935121
31.861406444
30.762901496
15.998900201
18.875384018
17.796242151
20.059068434
17.150372977
19.520210166
22.838277513
21.448412749
23.013539421
23.176132138
24.183281257
66.725811363
67.230829851
68.450716972
66.301114827
66.606999000
63.173598248
63.015059003
63.992668518
64.135944000
61.756692791
61.601916000
70.992773130
71.379830714
72.050988013
69.680606054
70.699555285
69.406357423
72.019260697
70.745339438
70.682599503
69.467895125
69.724714341
69.614149736
68.772705076
69.450860817
67.654950354
66.899907971
68.434849042
67.645117964
67.999688817
67.060316220
66.038902686
64.752682694
64.249730320
63.973071911
64.439728720
65.624630893
66.385964949
66.001017221
64.368131256
68.569901127
66.979047653
67.216536069
65.326548637
62.364478166
60.962804717
63.417926757
62.971692423
62.167506953
49.560577269
48.512053116
48.391819023
47.801645686
46.924000000
51.626585547
50.313587506
49.706838173
51.642252000
49.904883312
49.210267000
55.222100805
56.610938025
54.411627674
55.117769936
52.976510893
52.387730903
52.329455069
54.613738417
53.159638568
53.358486133
54.433940646
55.777374381
54.419919706
54.983468680
55.331632884
55.841778713
56.391012672
56.886543873
56.981773468
57.513881893
57.793092997
58.339036767
58.668194564
58.493790827
58.096770102
57.543889148
57.419241473
60.441270803
62.150847692
59.166930017
59.781949995
49.324954094
47.964201332
51.788096137
50.466662725
56.954529043
58.660278388
57.433072827
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
21.968315487
21.131969104
22.296855490
22.722076751
17.082953432
18.282017393
14.868800464
14.307699057
15.756641593
18.577844926
17.715448533
18.465668086
17.638623195
19.675020664
20.438092463
20.393642805
21.258464040
23.949323417
22.654038070
22.756984483
25.315458395
25.083730343
25.631185421
23.876057967
23.836308899
25.512952902
24.044135726
24.662265224
23.628966561
25.260894383
26.826523514
26.391188990
23.070920085
23.177509128
24.917726624
24.080724919
24.788142556
22.783667692
22.480222370
21.196159411
19.337595456
17.256381060
18.645779260
17.130714794
20.728196778
22.825998702
23.498938391
22.034406235
19.597235084
61.602504452
61.937337108
60.795129546
58.131059396
57.785993039
59.025203081
58.248012699
59.853375033
61.578805649
61.095949826
62.610984277
60.789531756
62.347497390
63.373264519
61.798797440
61.937595698
65.240064158
64.315194841
65.517012236
66.974647772
66.676675936
65.283842544
67.013634780
66.861150556
68.794999805
69.064367468
70.434230245
69.353992058
69.183465429
68.929436100
69.472344048
69.359755110
68.487768591
69.039467402
69.123359355
71.268758390
71.466440065
72.770105456
71.421983490
70.271666429
70.412064169
71.164883224
73.615872636
72.878810833
74.175166211
74.141134293
72.133306725
70.125065802
69.167195454
55.519777385
57.601858798
59.496155816
57.257012792
59.138408489
59.043784049
59.093224125
59.292390774
59.025958828
58.673980870
58.856813160
61.206178498
61.309153851
60.081173738
60.191079583
62.683958141
61.958549044
61.176876079
59.319658057
60.301847310
60.308207885
59.256091802
57.741359541
57.617618344
59.452949068
58.955468168
57.746427888
56.652416298
52.320928831
52.704181431
51.134218323
49.436127251
50.044464627
47.885330299
47.973451844
47.233746171
48.834874611
48.778246017
49.859002137
48.008129625
49.031099258
49.554712318
50.735060352
51.242437692
51.886631026
53.193090413
54.456931725
54.529059470
53.512509581
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
O
H
O
C
H
H
H
H
H
H
H
H
H
H
H
17.738530380
20.433586958
18.800967263
16.863184238
19.621625356
17.475894857
16.571239873
15.091491693
16.172506076
33.282068591
34.802820063
28.071674953
27.325964922
32.429577629
28.995785747
30.218345634
31.374677395
26.456225200
25.089760187
28.444449460
28.642564227
26.903359730
24.321793751
24.406149374
23.573840831
25.033228782
32.431853802
32.273908652
17.805069567
16.799331941
15.871046960
16.849535000
22.479645970
22.145641407
22.982507000
18.548727025
21.957957800
20.601667000
20.992916612
22.055332129
33.334852547
31.919450028
23.073901009
14.634821341
17.945662409
69.961903943
73.647113439
74.989926405
73.431723793
71.273026520
69.886292957
70.691868482
69.974205057
73.028655595
66.279289411
68.747351870
63.405097418
64.892373289
63.771489822
68.957914186
68.637714107
69.237613067
72.902622354
73.887203658
73.270837863
74.295870087
75.623471940
75.494384484
74.501887421
76.778246752
77.493915021
68.678582249
70.756243646
68.397856411
64.674444356
65.200724700
63.603478000
66.129784626
65.894565812
66.274601000
64.715109587
65.468877866
66.337713000
67.688065055
64.817006549
68.404237196
67.018993720
69.169245698
64.859006964
66.948779314
52.060429574
56.239485666
54.829002318
54.339924407
56.675394756
55.628192895
54.356615530
56.144882179
57.725259067
56.502594167
55.348241684
59.231385823
58.763133537
58.243298616
56.629129742
53.628388092
52.411725890
51.734094430
51.332326799
50.853856644
49.453074111
48.820241178
50.162781418
48.684225925
48.198869323
48.912966881
57.264046977
54.334798895
61.795300008
49.033949350
48.413368754
49.253524000
48.714302841
47.343233169
46.742711000
50.030492470
49.223331701
45.892815000
46.958714262
47.153192080
53.414193530
54.764747228
57.280881859
60.513068393
59.223140218
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state K144A 4Fe-4S quantum cluster
model in chain-A.
↓
199 atoms
Fe
-0.144539720
Fe
-0.245547735
Fe
2.157733280
Fe
0.656450272
S
1.549709570
S
1.828131163
S
-1.510088508
S
0.538766374
S
-1.213728320
S
4.310417364
S
0.871519588
S
-1.026377570
N
2.812448972
C
2.637610287
C
3.905498942
N
4.779311669
O
4.026239451
C
1.514590873
C
0.145776715
C
-0.834718271
N
-2.146327437
C
-3.194582402
N
-3.138723046
N
-4.351057333
H
2.384682756
H
1.457685146
H
1.800434369
H
0.203181950
H
-0.211348637
H
-0.949835090
H
-0.445716832
H
-2.260023283
H
-2.384065790
H
-4.006171862
H
-4.312719592
H
-5.047549491
H
2.538318941
C
5.977111000
H
6.869350000
C
2.007541964
H
1.743299000
C
2.693467027
O
2.561421422
C
2.826115350
0.196975706
0.093471428
0.128683596
-2.058096363
-1.346762810
-0.958191792
-1.179246970
1.792018095
1.210444745
0.743907225
-4.257752479
0.973455188
-1.144235117
-2.544295466
-3.231916668
-2.443796413
-4.460180023
-2.708143239
-2.228411752
-2.255721559
-1.707827787
-2.364724204
-3.663138384
-1.702824618
-3.071675198
-3.766530257
-2.176120872
-1.202523485
-2.865253741
-3.266219306
-1.636691219
-0.709360962
-4.251659661
-4.142618411
-0.690154037
-2.136519163
-0.441901919
-2.929906000
-2.636805000
5.096863435
6.022431000
5.355877522
4.601486240
4.170938441
↓
↑
-0.081662473
2.608586257
1.357114349
1.144667721
3.028719584
-0.555516387
1.231168536
1.274793729
4.387821635
1.617286379
0.497020769
-2.039924457
8.372553786
8.058889141
7.521788363
6.879809248
7.657665862
7.036775623
7.510617751
6.340420228
6.658423801
7.142315038
7.434378422
7.321199643
8.984167769
6.787462466
6.122352578
7.890415999
8.329337480
5.948266559
5.531832822
6.511711330
7.093097071
7.651859486
7.382939658
7.918862695
7.687048409
6.238907000
6.801957000
0.558304007
0.036064000
1.881632020
2.855244407
-0.361236480
↑
C
N
C
N
C
H
H
H
H
H
H
H
N
C
H
C
H
C
N
O
C
H
H
H
C
C
N
O
H
C
H
H
H
H
H
C
C
N
O
C
H
H
H
H
N
C
C
N
O
4.201817764
4.503115478
5.820323090
6.376502281
5.406405184
1.071523408
2.255141145
2.917631749
3.831413838
5.643312777
6.335950163
7.379278827
1.856169819
1.539951000
1.476316000
0.600139221
0.982050000
1.749542100
2.568122038
1.941730204
-0.458234231
0.137540461
-1.283220901
-0.050034124
3.741267468
4.634344640
4.848478381
5.222167206
2.428199248
4.622262421
3.441021090
4.597010178
5.655146174
5.323203000
0.913280561
-0.076931442
-1.121015034
-2.225150430
-1.005769704
-0.351988884
-0.137435575
-0.347090072
-1.346357534
-3.034079000
-2.441811576
-1.392344028
-0.674636879
-0.971267746
0.146511327
4.604468308
5.650132570
5.763195191
4.814275844
4.080900984
4.580277634
4.026576917
3.196429434
6.246312474
3.242209284
6.486869947
4.663004000
2.444883833
3.763438000
4.465197000
3.064758941
4.110914000
2.065825801
2.101465341
1.276341840
2.896883357
2.935474359
3.583046328
3.177450482
1.243701718
1.377105116
2.629101260
0.405332353
2.814222611
1.590073606
0.196938488
2.672489178
1.330713512
2.690085000
-5.169970008
-5.323146549
-5.077908007
-5.834260580
-4.160725881
-4.278101468
-6.338515473
-3.293242921
-4.443590117
-5.540736000
-1.116416310
-0.233881081
-0.705064787
-1.935288262
0.045820480
-0.714376983
-1.565363840
-1.718525440
-0.976881696
-0.346048050
0.777154490
-1.282882422
0.116167138
-2.042071826
0.285402693
-2.328285207
-0.894333609
-6.992414952
-6.462627000
-7.300786000
5.650834713
5.606559000
5.613363571
4.534639949
6.551524102
4.552823618
6.633947941
4.778175521
3.582130960
4.437453430
5.664618920
6.087704335
6.152798605
3.823682266
3.220573579
4.397922236
3.058796929
3.473321348
6.970952000
-2.390702619
-1.951690960
-3.012873410
-2.943215550
-3.844589613
-0.865378165
-1.547109648
-1.333853327
-0.437128310
-3.483254000
-4.092100095
-4.539824077
-5.813125889
-6.242354397
-6.355420715
C
H
H
H
C
H
C
C
H
N
O
C
O
C
H
H
H
H
H
H
H
C
H
C
O
C
C
C
N
C
N
N
H
H
H
H
H
H
H
H
H
H
H
H
N
H
C
H
C
-0.362604066
-1.853385630
-0.027892634
0.502485696
-0.358250000
-1.111312000
1.024796395
-0.381202231
1.466691000
-0.964802344
-0.942383583
1.172779600
0.291614764
2.627036458
1.609254699
0.875399507
0.373810455
2.784264283
2.895701862
3.297886321
-1.938787000
3.080778000
3.027971000
2.910362646
3.701720338
4.469375333
4.686497028
4.127174020
4.835645746
5.963889004
6.605076718
6.446010429
2.282101537
4.654404854
5.215880609
5.761408753
4.231874553
4.153753723
3.080407287
4.516033838
6.524375933
7.481669190
5.838097113
7.270026692
3.489086753
4.046672000
6.118509144
5.273395434
2.585915467
-0.033679726
0.723224959
-1.008527346
0.467601396
-2.570636000
-2.639925000
-6.172020935
-6.384679047
-7.130947000
-7.529615461
-5.587328642
-5.394337274
-6.012699886
-5.457299971
-5.700846682
-4.349926529
-5.486204988
-4.852928015
-6.496951344
-5.087420075
-7.684525000
0.711402000
0.637566000
2.171393095
3.048051651
0.177051292
-1.299821229
-2.300370970
-2.284549859
-2.932761345
-2.623032971
-3.884251711
0.105254636
0.354928890
0.762458782
-1.493052406
-1.522736934
-3.313616872
-2.104153084
-1.619542197
-1.680822787
-3.092495367
-4.361924103
-4.395147250
6.436760978
6.552932000
-2.534096495
-2.908031213
4.280238845
-3.425707863
-4.798060133
-3.060397943
-3.857322660
-7.389921000
-8.181831000
5.297517751
5.806496618
5.085305000
5.418589463
6.576674390
3.994759458
3.044233304
3.537865294
6.088111931
4.139264043
2.205674506
2.641734438
3.310488239
4.319697343
5.661722000
-8.141230000
-9.232461000
-7.772868584
-8.178335673
-7.745356388
-8.093367596
-7.084140384
-5.801788745
-5.503817007
-4.368911530
-6.297016668
-7.713198256
-6.678917772
-8.290637508
-8.199733207
-9.068309010
-7.494236335
-6.845440963
-5.106593289
-4.004502512
-4.165003039
-6.951950882
-5.997911872
1.945488460
2.783092000
4.775778759
4.188356485
-5.444914452
C
C
N
C
C
C
C
C
C
C
H
O
C
O
H
C
O
H
N
C
N
N
C
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.992717679
2.144371628
2.832423922
4.151366219
4.294876313
5.222000959
6.473313804
6.649582049
5.580074897
0.158347059
0.736894000
1.034559585
-3.756400938
-4.283111423
-4.321425000
3.248216378
3.610123990
3.237939000
5.685215154
6.766777249
6.750749884
7.846940163
9.095943897
9.027971000
8.816379000
4.612130844
1.284083682
4.233718334
-2.365183072
-1.720322792
-0.549770154
3.643926842
7.039823407
6.174983520
0.554755741
2.156375963
3.470308856
1.084382694
2.431871601
5.085466136
7.333526225
7.648087699
5.744352492
-2.167188410
0.455217907
-0.695761942
0.456937801
10.002674909
8.272462401
3.267220451
2.612553461
1.675128546
1.703362000
2.708085452
0.941404091
1.198596663
2.200888091
2.954390434
-3.976985130
-4.386578000
-3.981657081
-0.834810256
0.154115613
-1.617047000
-0.686214639
-1.372738534
0.408880000
4.785242694
4.063506617
3.408908253
4.081347616
3.361601094
1.861262000
1.617659000
-1.442774718
1.665761139
3.389650067
-6.506084481
-2.433186065
-8.118290737
7.058266068
-2.979067182
-1.451312262
3.687474532
5.175167392
4.609072661
2.741203564
1.074051860
0.189464251
0.631511831
2.389255499
3.737599132
-1.950774367
-1.929121790
-4.645683488
-3.963778429
1.427178222
1.365597725
-4.416426348
-3.559210914
-2.833554458
-3.189666080
-4.185933042
-2.724136617
-3.262841848
-4.235542875
-4.693699016
-7.040865947
-7.879897000
-5.913029688
-4.116946768
-4.634513775
-3.604721000
9.555140726
10.517978776
9.561120000
-9.711400030
-9.381649429
-8.211676521
-10.149707634
-9.894271082
-10.151451000
-11.197832000
6.827446266
-6.680593046
5.834880350
-2.197062774
-5.773464770
4.706218661
1.161887032
4.380254406
4.653060197
-5.996679592
-4.972401173
-5.994986280
-3.402638150
-2.121947347
-1.953212533
-2.913523220
-4.623704406
-5.425912713
-3.579479008
-7.727853337
-6.865104064
-5.119533083
-9.898667945
-9.535628347
H
H
H
H
H
H
H
H
9.837803924 3.826521280
9.415147566 3.566252540
7.786238656 4.629070670
5.609690109 5.121476157
4.805411933 4.514698268
7.504958602 2.774477414
5.832743423 3.164225281
5.939707193 -4.023057639
-10.548967810
-8.865440017
-11.003459183
-10.665836917
-9.276445254
-7.988398224
-7.848882242
6.306787206
Cartesian coordinates of the optimized Fe1 Fe2 Fe3 Fe4 state K144A 4Fe-4S quantum cluster
model in chain-B.
↓
238 atoms
Fe
19.268335560
Fe
17.376200400
Fe
19.766953580
Fe
17.883098382
S
17.673193087
S
20.118713271
S
17.008582048
S
19.513948877
S
15.588360345
C
15.122855075
S
20.388074653
C
19.905033813
S
21.488302176
C
20.971145355
S
17.122956274
C
18.474698112
H
15.379391155
H
14.030627707
H
20.815330046
H
19.190256431
H
20.462644676
H
20.241797878
H
18.832152294
H
19.318835026
C
24.644002000
C
24.906773378
N
26.343827156
C
26.910555091
N
28.226394238
N
26.177129048
H
25.141613000
C
21.415000000
67.144381298
65.191752674
64.805121281
66.469388509
64.184435227
66.940447334
67.378250176
65.059175055
64.768911827
66.335184712
68.751871693
68.895320328
63.502554556
63.178838350
67.241353475
67.264269791
67.164073946
66.318570800
69.190783935
69.718486442
62.208875257
63.939205804
68.288094812
66.660318402
76.620997000
75.402352710
75.240257367
74.297229615
74.091161872
73.547221170
76.554792000
71.340995000
↓
↑
56.343164714
56.405553394
55.143483781
54.085820800
54.414749976
54.315918322
55.889465724
57.288876755
57.785182040
58.635127693
57.490744061
59.250410433
54.398534654
52.674317423
52.084506078
50.851460825
57.976575049
58.735843029
59.780768421
59.362621245
52.649730366
52.392206277
50.740240193
51.197157226
48.376996000
49.235949019
49.451219081
50.203496348
50.131487156
51.034642944
47.403845000
47.975998000
↑
C
O
C
C
C
N
C
N
N
H
N
C
C
C
C
C
N
C
C
C
C
C
H
C
C
O
C
C
N
C
C
N
H
N
H
N
C
C
O
C
C
C
N
C
N
N
C
O
H
20.195200684
20.134925473
22.655200874
23.955855826
24.073049617
23.976558624
24.943080068
24.635967322
26.162204993
21.618741000
19.205745792
17.991000000
18.201255198
19.048099105
18.797272579
20.309476231
19.824245946
20.770104581
21.103967204
21.981723051
22.308051888
22.749403703
17.633684000
15.749467719
16.058147820
16.212321585
17.019617362
17.888570603
17.583671332
19.107303824
18.564434418
19.493116306
15.078911000
16.174890324
16.562673000
19.506560802
20.331197463
21.790682546
22.667427377
19.705806352
18.336027276
17.752337249
16.440764388
16.193717194
14.921931571
17.176354473
19.406011179
20.028400718
18.677061000
72.132028118
73.365842520
71.705696386
71.123590637
69.600463261
69.054357099
69.120638496
68.767933963
69.518660224
71.609153000
71.406310231
71.973996000
72.681850965
71.930432239
70.673230403
72.327415386
70.266305960
71.250873887
73.480574133
71.288630968
73.524526717
72.436695803
72.744707000
70.922586945
70.978330202
69.951399732
70.939399277
72.134051644
73.384454557
72.268424239
74.243715034
73.572769573
71.721456000
72.206081455
72.275889000
63.814494152
62.828089263
63.270866326
62.398969587
62.451606552
61.780271805
61.635318546
60.997339363
59.693403073
59.271464958
58.791865381
63.955346110
63.315114803
64.732842000
48.405828949
48.257903534
48.796479825
48.241108684
48.282420309
49.635245442
50.558977740
51.827044291
50.248430335
46.934073000
48.957418529
49.510997000
50.869631264
51.844791298
52.328770369
52.398977908
53.140327275
53.205110481
52.312357545
53.890733725
52.998319992
53.773885820
48.820089000
56.272169046
57.759283375
58.433895127
55.400362695
55.533036591
55.032655396
56.129599903
55.297738991
55.967180366
55.939196000
58.306100022
59.244498000
62.711775634
62.047332983
61.814169512
61.709296399
60.701279607
60.789273386
59.383474448
59.349718513
59.291891770
59.182193757
59.329195539
64.042509365
64.899383125
64.291576000
N
C
C
H
H
C
C
O
C
O
C
H
N
H
C
C
H
O
N
C
C
O
N
H
N
C
C
O
N
C
C
C
O
H
N
H
S
O
O
O
P
O
O
O
O
C
C
O
C
22.004058865
23.289001000
23.268299359
24.296234680
23.715284000
22.355536709
21.371833341
20.212604975
22.118574690
22.171369774
23.175892396
23.378128000
21.824966527
21.170100000
15.698730475
17.198516304
15.420306000
17.726136042
17.914856235
19.342178353
19.713146166
20.766501577
18.930604769
19.238077000
17.829743130
18.020176558
19.033561741
19.167880633
19.744212900
20.853000000
22.100927472
21.378623570
20.913448522
22.652466000
21.152661817
20.434047000
26.848492709
27.133824506
26.313124623
26.141831903
28.821577553
28.251156993
28.605117437
28.417664995
30.441160222
31.071300456
32.085008011
31.471411385
33.246374087
64.589300681
65.171998000
66.057575456
66.364725173
65.719872000
60.399580072
59.258929395
59.340567636
61.537823250
60.958557862
62.617795101
60.073913000
58.148889074
57.394184000
66.570481558
66.486829744
67.594069000
65.597504770
67.513152822
67.641586399
67.752435049
67.262236805
68.509635210
68.583743000
65.335658273
66.766434205
67.251511232
68.469537292
66.309584508
66.607000000
63.185044188
63.025410388
64.003482695
64.135944000
61.765348087
61.601916000
70.856337703
70.999789126
72.067043438
69.611478354
70.594041234
69.337871672
71.928872769
70.678661125
70.475598278
69.235579773
69.522445452
69.492339227
68.545846845
61.677818634
61.339999000
60.101251259
59.870110298
62.186842000
58.484156247
58.348229550
58.786883207
57.499826623
56.183195250
57.682854593
58.312272000
57.743975864
57.563116000
60.035213486
60.234137165
60.325163000
60.914687926
59.712642473
59.934360442
61.409090946
61.836530079
62.179107174
63.137216000
49.499633081
49.474825360
48.429659970
48.247890594
47.800102992
46.924000000
51.632789425
50.315592149
49.704706734
51.642252000
49.910684521
49.210267000
54.854352391
56.291598851
54.218868309
54.508872411
52.641765229
52.032641458
51.981937562
54.259597199
52.857467028
53.248702719
54.349889858
55.677956878
54.426850199
O
C
O
C
N
C
N
C
C
N
N
C
N
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
34.370824593
32.710342265
33.755303064
31.915223798
30.749311724
29.458935478
28.682183480
29.519276995
29.315611216
28.115416889
30.367810360
31.553324337
31.864054190
30.805632684
15.254157200
18.913097300
17.437025712
19.858258545
17.066092265
19.525155362
22.802839688
21.442229109
22.989346948
23.167501596
24.171280105
21.929257150
21.122617701
22.296375008
22.737206260
16.944061514
18.139311965
14.727908974
14.182796801
15.632785631
18.433163678
17.609289757
18.432224026
17.641838776
19.636504263
20.394859072
20.394489565
21.235569153
23.963453291
22.866337384
22.668868541
23.697165375
25.336658970
26.920942469
26.456651422
69.234223939
67.492421303
66.732957232
68.356571305
67.633418974
68.079537586
67.235003499
66.184311844
64.966721734
64.577153871
64.133873100
64.485727067
65.601381944
66.417163270
65.872680403
64.413424254
68.232083714
66.754628972
67.226387796
65.336617561
62.364470619
60.975211288
63.462004920
62.987378814
62.210733999
61.687692977
61.964416761
60.777167237
58.112428854
57.826593137
59.055868579
58.300714133
59.918935688
61.612180485
61.090176383
62.623777688
60.797822850
62.375566582
63.356397424
61.773934080
61.945643153
65.241866955
64.327469683
65.519499122
66.954401264
69.004449828
68.946697681
69.513677088
69.486978738
54.982787387
55.386625224
55.959428305
56.381511586
56.936252344
57.082655916
57.733404410
58.046562771
58.721688347
59.186313620
58.875096891
58.365948920
57.698379131
57.568786497
60.750034903
62.139604390
59.175717585
59.567759677
49.202882584
47.983620130
51.814047321
50.476369262
57.014787963
58.710974317
57.463938061
55.556423177
57.664151713
59.507728762
57.304575525
59.117846150
59.140506702
59.407843451
59.438748277
59.338315107
58.727625929
58.946813785
61.267861590
61.393177072
60.088309657
60.198264903
62.691973931
61.803947762
61.169681073
59.238377227
60.260547800
52.133418529
52.548448499
50.960107399
49.278801539
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
O
H
23.203204918
23.270554952
25.007186601
24.081255158
24.796256626
22.744256398
22.515944292
21.209118403
19.307063665
17.251498549
18.662864923
17.202409904
20.788748700
22.927669554
23.709715639
22.311841051
19.877178967
17.955430142
20.366156165
18.596630837
16.744559461
19.720913372
17.628504317
16.705030145
15.239003232
16.159769854
33.307165372
34.930766392
28.082794811
27.368959821
32.363837443
29.143331855
30.331038610
31.589874952
26.684752216
25.294870032
28.615237590
28.731722530
26.960514644
24.410774482
24.509833133
23.566870552
24.989981441
32.537888604
32.471662490
18.010977058
16.761835559
15.808328421
16.849534000
68.427163074
69.131315438
69.276099497
71.423931635
71.557754140
72.796784960
71.408632299
70.269473611
70.397836413
71.173740436
73.653430961
72.886914875
74.328282499
74.416594788
72.487554389
70.452915042
69.362267098
70.018823830
73.974746600
75.284332741
73.632437283
71.542807499
70.071018872
70.833144714
69.974353359
73.047043112
66.156114308
68.540948926
63.780241061
65.254732917
63.776381768
69.030700268
68.505894801
68.845058814
72.928318938
73.909402143
73.322328691
74.388581146
75.728652991
75.533860872
74.502784224
76.714097620
77.528429719
68.654802716
70.538585925
68.833429606
64.671117343
65.159833194
63.603479000
49.831085559
47.713078880
47.806962717
47.191178557
48.794414483
48.796339852
49.842905215
48.013922386
48.977617033
49.585479288
50.674376909
51.281171850
51.710128786
52.937618583
54.275799488
54.502282576
53.595387110
52.166420092
56.299370960
55.020442349
54.514300863
56.636765742
55.643988506
54.356913738
56.086542819
57.741976754
56.638239849
55.391979798
59.814290102
59.259090212
58.514240442
56.683944532
53.581991329
52.366663014
51.668008408
51.372095923
50.684336823
49.303544660
48.808997206
50.204072105
48.754032075
48.203735255
48.884949239
57.235180427
54.211441406
61.908446685
49.022724195
48.410500693
49.253524000
O
C
H
H
H
H
H
H
H
H
22.487007801 66.156084924 48.716479416
22.145156163 65.901954774 47.349428065
22.982507000 66.274601000 46.742710000
18.516693810 64.779521484 50.000993534
21.963534595 65.506167916 49.237252647
20.601666000 66.337713000 45.892815000
20.992600996 67.688935123 46.954401222
22.059073998 64.821562755 47.175536770
33.502150657 68.115238447 53.451532269
32.005326545 66.850504179 54.837810281
Supplementary Figure 1
Supplementary Figure 2
normalized XAS cross-section (cm2g–1)
350
– APSR
– APSR + APS
A
300
250
200
150
100
50
0
7090
7100 7110 7120 7130 7140 7150 7160
energy (eV)
k3-weighted EXAFS
10
– APSR
– APSR + APS
B
5
0
–5
–10
FT Magnitude
0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
5
10
15
k (Å-1)
– APSR
– APSR + APS
C
0
2
4
R (Å)
6
8
10
Supplementary Figure 3
– APR
– Ferredoxin [4Fe-4S]
– Ferredoxin [3Fe-4S]
χ (k) x k3
10
5
0
-5
-10
0
2
4
6
8
10
k (Å )
12
14 16
FT Magnitude
-1
2.0
– APR
– Ferredoxin [4Fe-4S]
– Ferredoxin [3Fe-4S]
1.5
1.0
0.5
0.0
0
1
2
3
4
R + ∆ (Å)
5
6
7