Diversity of Chemical Mechanisms in Thioredoxin Catalysis
Raul Perez-Jimenez1, Jingyuan Li2, Pallav Kosuri1,3 Inmaculada Sanchez-Romero4, Arun
P. Wiita1,5, David Rodriguez-Larrea4, Ana Chueca6, Arne Holmgren7, Antonio MirandaVizuete8, Katja Becker9, Seung-Hyun Cho10, Jon Beckwith10, Eric Gelhaye11, Jean Pierre
Jacquot11, Eric Gaucher12, Jose M. Sanchez-Ruiz4, Bruce Berne2, and Julio M.
Fernandez1
1
Department of Biological Sciences, 2Department of Chemistry, 3Department of
Biochemistry and Molecular Biophysics; 4Facultad de Ciencias, Departamento de
Quimica Fisica, Universidad de Granada, 18071, Granada, Spain; 5Graduate Program
in Neurobiology and Behavior, Columbia University, New York, NY 10027, USA;
6
Estacion Experimental del Zaidin, CSIC, 18008, Granada, Spain; 7Medical Nobel
Institute for Biochemistry, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, SE-171 77, Stockholm, Sweden; 8Centro Andaluz de Biología del
Desarrollo (CABD-CSIC), Dpto. de Fisiología, Anatomía y Biología Celular,
Universidad Pablo de Olavide, 41013, Sevilla, Spain; 9Interdisciplinary Research
Center, Justus-Liebig-University, D-35392 Giessen, Germany; 10Department of
Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115;
11
Nancy University, IFR110 GEEF, UMR 1136 Interactions Arbres Microorganismes,
Faculte des Sciences, 54506 Vandoeuvre Cedex, France; 12Georgia Institute of
Technology, School of Biology, Atlanta, GA 30332
1
Thioredoxins belong to a family of oxido-reductase enzymes present in all organisms
that catalyze thiol/disulfide exchange reactions reducing target disulfide bonds in
proteins. By applying a calibrated mechanical force to a disulfide bonded substrate,
the chemical mechanisms of Trx catalysis can be examined in detail at the single
molecule level. Here we use single molecule force-clamp spectroscopy to explore the
chemical evolution of Trx catalysis by probing the enzymatic chemistry of eight
different thioredoxin enzymes from four different kingdoms. While all Trxs have
developed a characteristic Michaelis-Menten enzymatic mechanism that can be
detected when the disulfide bond is stretched at low forces, two different chemical
behaviors distinguish bacterial from eukaryotic-origin Trxs when high forces are
applied. A computational analysis of Trx structures indentifies the evolution of the
substrate binding groove as an important factor controlling the chemistry of Trx
catalysis. Our results demonstrate the ability of force-clamp spectroscopy to identify
the diversity of chemical mechanisms in Trx enzymes, offering a novel approach to
understand the chemical evolution of this enzyme.
2
Introduction
Enzymes are exceptional catalysts able to accelerate reaction rates by several
orders of magnitude 1,2. However, little is known about how enzymes developed their
chemical mechanisms to obtain high reaction rates and specificity. The mechanisms of
numerous enzymatic reactions have been studied using protein biochemistry and
structural biological techniques like X-ray and NMR3-5. These studies have been useful in
identifying many structural features and conformational changes necessary for the
catalytic activity of enzymes. However, the dynamic sub-angstrom scale rearrangements
of the participating atoms necessary for catalysis cannot be detected by these techniques.
Recently developed single molecule techniques have shown promise in uncovering the
dynamics of enzymatic activity at a length scale that was previously impossible to
observe. For example, both optical tweezers and fluorescence techniques have been used
extensively to detect the motions of molecular motors, a large class of ATP consuming
force generating enzymes6,7.
In this work, we demonstrate the use of single molecule force-clamp spectroscopy
techniques to investigate the chemical mechanisms of catalysis of a broad class of
reducing enzymes called thioredoxins. These oxido-reductases are present in all known
organisms from bacteria to human. Thioredoxins posses a highly conserved active site
(CXYC) that catalyzes the reduction of target disulfide bonds, both inside and outside
cells, regulating a multitude of cellular processes8. The activity of thioredoxin enzymes
has been measured by monitoring the turbidity of solutions containing insulin, which
readily aggregates upon reduction of its disulfide bonds. Another approach has been
through the use of Ellman's reagent DTNB, where upon reduction by the enzyme
generates products that can be readily detected with a spectrophotometer8. While highly
effective in monitoring the overall activity of thioredoxins, these methods do not probe
the chemical mechanisms underlying the catalytic activity of these enzymes.
Recent single molecule force-spectroscopy experiments have revealed that the
application of a mechanical force to a substrate disulfide bond can regulate the catalytic
activity of thioredoxins9, revealing at least two distinct chemical mechanisms of
reduction that could be readily distinguished by their sensitivity to an applied force9. A
simple form of catalysis corresponded to a straightforward SN2 type chemical reaction
characterized by an exponential increase of the rate of reduction with the pulling force.
An additional chemical mechanism of reduction was characterized by its rapid inhibition
by a force applied to the substrate disulfide bond. This chemical mechanism is unique to
disulfide bond reduction catalyzed by thioredoxin enzymes and was explained by a
Michaelis-Menten type reaction where the binding of the enzyme to the stretched
polyprotein and the subsequent structural organization of the participating sulfur atoms
precede the chemical reaction.
Thus, the single molecule reduction assay that we have developed is able to
distinguish the chemistry of a simple SN2 reaction from more elaborate pathways for the
reduction of disulfide bonds which are unique to thioredoxin enzymes. It is of interest
then to consider the application of these sensitive single molecule techniques to study the
evolution of chemical mechanisms in this family of enzymes. The simplest evolutionary
hypothesis is that the ancient forms of thioredoxin had capabilities that were only
comparable to those of simple reducing agents. Evolutionary pressures then drove the
3
enzymes towards developing unique and more efficient mechanisms of reduction. As a
first step towards understanding the evolution of thioredoxin chemistry, we have applied
the single molecule reduction assay to examine the chemical mechanisms of reduction of
a sample of eight extant thioredoxin enzymes covering four kingdoms. We find that
while thioredoxins of bacterial origin posses both chemical mechanisms of reduction,
those of eukaryotic origin show only the enzymatic mechanism, having eliminated the
non-specific SN2 chemistry. These changes in the catalytic chemistry correlated well
with a deepening of the binding grove observed in thioredoxins of eukaryotic origin.
These results identify evolution of the binding groove as an important structural
adaptation controlling the disulfide reduction chemistry in the thioredoxin family of
enzymes.
Results
Selection of thioredoxins from different species
In order to investigate the variety of catalytic mechanisms developed by Trx we
have selected of a set of Trxs belonging to a representative group of species from
different kingdoms. Thioredoxin is highly distributed existing virtually in all extant
species. In addition, the existence of a second paralogous gene encoding Trx2 seems to
be common in animals, protists and Gram-negative bacteria10-14. In protists and animals,
Trxs1 is located in the cytosol whereas Trx2 is present in mitochondria10,14,15.
Interestingly, mitochondrial Trx2 from mammals have been shown to have higher
similarity with E. coli Trx1 than with cytosolic Trx1 from mammals12, suggesting the
bacterial origin of mitochondria16. In the case of plants, a rich variety of Trx genes can be
found enconding more than 20 different types of Trxs17 that are classified into six
isoforms: Trxf, h, m, x, y and o. The f, m, x and y forms are found in chloroplasts; h forms
are cytosolic Trxs and o forms are found in mitochondria. We have included both human
cytosolic and mitochondrial Trxs (Trx1 and Trx2, respectively) from animals; poplar
Trxh1 (featuring a CPPC active site), poplar Trxh3 and pea chloroplastic Trxm from
plants; E. coli Trx1 and Trx2 from bacteria, and finally we have chosen Plasmodium
falciparum (malaria parasite) Trx1 from protists.
A sequence alignment of the Trxs of interest shows that the residues around the
active site are highly conserved (Fig.S1). The construction of a phylogenetic tree (Fig.1),
incorporating additional Trxs from the three domains of life, classifies E. coli Trx1 and
Trx2, human Trx2 and pea Trxm as bacterial Trxs (top branches in Fig.1) and human
Trx1, poplar Trxh1 and h3 and P. falciparum Trx1 as eukaryotic Trxs (botton branches in
Fig.1). The construction of a larger tree incorporating over 200 Trx sequences (not
shown) corroborates that the sequences used are widely distributed and they are
representative for the entire Trx family.
Force-dependent chemical kinetics of disulfide reduction by thioredoxins from different
species
Similar to our previous published work, we have used an atomic force microscope
in its force-clamp mode to study the chemistry of disulfide reduction by Trx9,18. Briefly,
4
we have used as a substrate a polyprotein composed of eight domains of the 27th module
of human cardiac titin in which each module contains an engineered disulfide bond
between positions 32nd and 75th (I27G32S-A75C)8 9. A first pulse of force (175 pN, 0.3 s) is
applied to the polyprotein which allows the rapid unfolding of the I27G32S-A75S modules
up to the disulfide bond. The individual unfolding can be registered as steps of ~10.5 nm
per module. After the first pulse, the disulfide bonds become exposed to the solvent
where the Trxs molecules are present in the reduced form due to the presence of Trx
reductase and NADPH (Trx system)8. A second pulse of force is applied to monitor
single disulfide reductions by Trx enzymes that are recorded as a second series of steps of
~13.5 nm per domain (Fig.2A and B). We have accumulated several traces per force (1550) that have been averaged and fitted with a single exponential with a time constant τ
(Fig.2C and D). We thus obtain the reduction rate at a given force (r = 1/τ).
We have applied our single molecule assay to obtain the force dependency of the
rate of reduction by the selected Trxs (Fig.3). From these data we can readily distinguish
three different types of force-dependencies. First, all tested Trx enzymes showed a
negative force dependency in the range 30-200 pN. Second, all Trx enzymes from
bacterial origin show that after reaching a minimum rate at around ~200 pN, the rate of
reduction increases exponentially at higher forces. Third, at forces higher than 200 pN,
enzymes from eukaryotic origin show a rate of reduction that becomes force independent.
We have proposed that the reduction mechanism observed when the substrate is
stretched at low forces (30-200 pN) is similar to a Michaelis-Menten reaction in which
the formation of an enzyme-substrate complex is determinant. Upon binding, the
substrate disulfide bond needs to rotate in order to achieve the correct geometry
necessary for an SN2 reaction to occur, i.e., the three involved sulfur atoms forming a
~180° angle9,18,19. This rotation causes the shortening of the substrate polypeptide along
the stretching force axis determined by the negative value of Δx12 in our kinetic model
(Table 1). This mechanism is rapidly inhibited as the force increases generating the
negative dependence of the reduction rate with the pulling force in all Trx enzymes
(Fig.3). While the absolute rate of reduction varies from enzyme to enzyme, the gross
characteristics of this mechanism of reduction is apparent in all of them.
According to the parameters obtained from the fitting to a simple MichaelisMenten type kinetic model (Table 1), we found that an extrapolation to zero force
predicts rate constants ranging from 1.2 x 105 M-1∙s-1 for poplar Trxh3 to 6.0 x 105 M-1∙s-1
for human Trx2. The value of Δx12, remains below 1 Å except for E. coli Trx2 and poplar
Trxh3 with values over 1 Å (Table 1). These high values of Δx12 represent a higher
rotation angle of the substrate disulfide bond for the SN2 reaction18. This mechanism is
unique to Trxs enzymes and it seems to be the result of evolutionary pressure toward
developing an efficient mechanism of disulfide reduction not possible with simple
chemical reagents20.
When forces over 200 pN are applied to the substrate, the Michaelis–Menten
mechanism is blocked and a second force dependent mechanism of reduction becomes
dominant. This is true in all types of Trx enzymes. In enzymes of bacterial origin this
high force mechanism (Fig. 3A) appears to be analogous to that of simple chemical
compounds such as cysteine, glutathione or DTT which reduce disulfide bonds through a
force-dependent SN2 thiol/disulfide exchange reaction with bond elongation at the
transition state20,21. We have incorporated this reaction in our kinetic model (k02),
5
obtaining a value for the elongation of the disulfide bond at the transition state of ~0.17 Å
(Δx02 in Table 1), similar to the values obtained when using cysteine as a nucleophile(~0.2
Å)20. In the case of eukaryotic Trxs this SN2 like mechanism of reduction is absent;
instead, the rate of the reaction becomes force-independent at higher forces (Fig. 3B).
This force-independent mechanism gives a constant rate of reduction that ranges from 0.2
to 0.4s-1 (inset in Fig.3B). We speculate that in enzymes of bacterial origin, the minimum
value of the reduction rate is also limited by this force-independent "floor" in the rate of
reduction which varies in the range 0.2 to 0.4s-1 (inset in Fig.3A). We have incorporated
this mechanism in our kinetic model in the form of a constant parameter λ (see supp
information and Fig. S2), that contributes equally to the reduction rate throughout the
entire range of forces (Table 1). An interesting possibility that may explain this force
independent chemical mechanism is a single-electron transfer reaction (SET) via
tunneling, a process that has been observed in enzymes containing metallic complexes4,22.
In addition, it has been suggested that SET are highly favored when steric hindrance
occurs23. To test whether an electron transfer mode of reduction would be force
independent we have investigated the kinetics of disulfide reduction under force by a
metal. Metals are able to reduce organic substrates by tunneling a single electron in a
reaction governed by the reduction potential (refs). We have studied the reduction of
disulfide bonds by Zn nanoparticles (diameter <50 nm), which has been shown to reduce
disulfide bonds in proteins24. In sharp contrast to all other reducing agents that we have
studied, the rate of reduction of disulfide bonds by Zn is force-independent (Fig. 5A).
These results support the idea that the force-independent mechanism is a barrier free
electron tunneling reaction that does not require the precise orientation for the SN2
reaction, however, further experiments including temperature and concentration
dependence would be required in order to fully confirm the SET mechanism.
Our results show that there are three distinct mechanism of reduction that operate
simultaneously in a thioredoxin enzyme. These mechanisms are identified by their force
dependency as illustrated in Figure 4. The most complex mechanism is characterized by a
negative force dependency and is unique to enzymatic catalysis by Trx (Fig. 4A,B). This
enzymatic mechanism of reduction is characterized by a Michaelis Menten reaction
between the substrate polypeptide and the binding groove of the enzyme, followed by a
rotation of the substrate disulfide bond to gain position for the SN2 reduction
mechanism9,18 (Fig. 4A,B). A much simpler mechanism is that of a regular SN2 reaction,
characterized by a rate of reduction that increases exponentially with the applied force.
This mechanism is well represented by nucleophiles such as L-cysteine (Figure 4A),
glutathione and DTT 9,20. In this mechanism the substrate disulfide bond and the catalytic
cysteine of the enzyme orient themselves with the pulling force, without needing a
rotation of the substrate disulfide bond (Figure 4C). We suspected that this mechanism
would be possible only if the Trx enzyme had a shallow binding grove that allowed many
other orientations of the substrate-enzyme complex. Finally, the third mechanism is the
force-independent barrier-free electron tunneling transfer mechanism illustrated by the
action of metallic zinc (Figure 4D). It is inevitable that if the disulfide bond gents close
enough to the thiolate anion of the catalytic cysteine, that electron tunneling will occur,
albeit at a very low rate.
Thus, comparing the data of Figures 3 and 4, it is clear that the main difference
between enzymes of bacterial and eukaryotic origin is the elimination of the high force
6
simple SN2 like mechanism of reduction and an enhancement of the rate of reduction
through the Michaelis Menten-SN2 reduction mechanism. We speculated that this drastic
change in the catalytic chemistry would be caused by changes in the structure of the
enzyme as it evolved. The most salient feature in the structure of Trx enzymes is the
binding groove into which the target first polypeptide binds, to be subsequently reduced
by the exposed thiol of the catalytic cysteine (Fig. 5A).
Structural analysis and molecular dynamics simulations of thioredoxin binding groove
In order to study the role of the structure in the chemical behavior of Trxs, we
have analyzed the structure of the binding groove of a set of bacterial and eukaryoticorigin Trxs. We have studied three eukaryotic-origin enzymes: Human Trx1, A. Thaliana
Trxh1 and Spinach Trxf, and three bacterial-origin enzymes: Human mitochondrial Trx2,
E. coli Trx1 and C. reinhardtii Trxm. From the X-ray or NMR structures we have
defined structural axes that allow us to calculate the depth and width of the binding
groove in the region surrounding the catalytic cysteine (Fig. 5A and supplementary
information for details). We found that eukaryotic Trx enzymes have binding grooves
that are several Angstrom deeper than those of bacterial origin (Fig.5B). By contrast, the
width of the binding groove remained the same (not shown). We explored the
consequences of a deepening binding groove using molecular dynamics simulations to
probe the mobility of a bound polypeptide (see supplementary information). For the MD
simulations we have considered a set of enzyme structures obtained with mixed disulfide
intermediates between the catalytic cysteine and a cysteine in the bound substrate. Such
structures capture the general disposition of the substrate in the catalytic site of the Trx
enzyme. We have used three eukaryotic complexes: Human Trx1 with the substrate
REF-1; Human Trx1 with NF-κB and barley Trxh2 with protein BASI, and two bacterial
complexes: E. coli Trx1 with Trx reductase and B. subtilis Trx with Arsc complex. In
order to compare among these structures, 13 residues of the substrates are taken into
account, with the binding cysteine always set to be the 7th residue. For the MD
simulations we have removed the substrate-enzyme disulfide bond to allow substrate
mobility. Fig. 5C shows that the shallow binding groove of bacterial Trxs allows the
substrate to be mobile. By contrast, the deeper grove found in Trx enzymes of eukaryotic
origin tends to freeze the substrate into a much smaller range of conformations.
Similarly, the measured distribution of the distances between the reacting sulfur atoms is
smaller and more narrowly distributed in the deeper binding groove of Trx enzymes of
eukaryotic origin than in those in the shallower grooves found in enzymes of bacterial
origin (Figure 5D). These structural observations suggest that a major feature in the
evolution of thioredoxin enzymes has been an increase in the depth of the binding
groove, increasing the efficiency of the MM-SN2 mechanism and eliminating the plain
SN2 mechanism of catalysis.
Discussion
Over the last 4 billion years the chemistry of living organisms has continuously
changed in response to atmospheric and biological phenomena. For instance, the increase
in the oxygen levels (~2.5 Gyr) provoked a chemical expansion25,26 that had tremendous
impact on the chemistry of enzymatic reactions especially those implying redox
7
transformations26,27. However, understanding how enzymes have adapted their chemical
mechanisms to evolutionary pressures remains a mystery in molecular biology. In this
work, we probe single molecule force-clamp spectroscopy as a valuable tool to examine
the evolution of Trx catalysis by studying the chemistry of eight Trx enzymes from four
different kingdoms.
We show that three different chemical mechanisms for disulfide reduction can be
distinguished in Trx enzymes by their sensitivity to an applied force (Fig. 4). While all
Trx seems to have developed an efficient Michaelis-Menten mechanism for disulfide
reduction (Fig.4A and B), only those of bacterial-origin are able to reduce disulfide bonds
throughout a straightforward SN2 reaction comparable to that of a simple cysteine
(Fig.4A and 5C). This SN2 reaction seems to have been eliminated in eukaryotic Trxs
due to steric hindrance in the binding groove. Instead a reduction mechanism that might
be explained by a single-electron transfer reaction via tunneling can be observed (Fig. 4A
and 4D). Thus, it appears that the mechanism of disulfide bond reduction by Trxs was
altered at an early stage of eukaryotic evolution.
The ability of force-clamp spectroscopy in separating the complex enzymatic
mechanism from more simple chemical reactions raises an interesting scenario towards
understanding the evolution of the catalytic chemistry of Trx. The simplest evolutionary
hypothesis is that reduction mechanisms similar to that of simple chemical compounds
might be primordial forms of disulfide reduction in Trx. However, the evolutionary
pressure forced the appearance of a more efficient reduction mechanism in Trx. We
identify the binding grove as an important factor that permitted the evolution of Trx
catalysis. The appearance of the hydrophobic groove allowed Trxs to bind the substrate in
a specific fashion, generating a stabilizing interaction that made the enzyme capable of
regulating the geometry and orientation of the substrate disulfide bond. This situation
originated the Michaelis-Menten mechanisms that clearly enhanced the chemical
capabilities of Trx over simple reducing agents. Indeed, mutations affecting the binding
region of E. coli Trx have a prominent effect on this mechanism while barely affecting
the SN2 mechanism (ref. 18). The emergence of eukaryotes gave rise to more complex
biological systems bringing a palette of new functions and targets28. It is tempting to
speculate that the deepening of the binding groove in eukaryotic Trxs (Fig.4A) seems to
have been an important structural adaptation that improved their interaction to specific
substrates. These structural rearrangements clearly had a consequence in the chemistry of
Trxs by sacrifying the sterically demanding SN2 reduction mechanism.
The evolutionary perspective that we infer from our enzymatic assay at the single
molecule level might be extended to the cellular level. For instance, both human
mitochondrial Trx2 and pea chloroplastic Trxm, show the force-accelerated SN2 reaction
typical for bacterial Trxs. These results are in agreement with the serial endosymbiotic
theory which states that both mitochondria and chloroplasts were once free living bacteria
developed from proteobacteria and cyanobacteria, respectively 16,29. In fact, human Trx2
and pea Trxm are grouped with proteobacteria and cyanobacteria , respectively (Fig. 1).
Although the endosymbiotic theory is nowadays generally accepted and well-supported
by phylogenetic studies30,31, our results represent an experimental and phylogenicindependent test at the single molecule level.
From a biological point of view, the SN2 reaction could be related to the ability of
bacteria to live under extreme situations in which elevated forces can be found such as high
8
pressures or extreme salinity conditions which may generate drastic changes in intracellular
osmotic pressures causing cells to swell or shrink32-35. Strikingly, Trx has been shown to
promote high-pressure resistance in E. coli36. Nevertheless, it is important to note that we
use force to restrict the orientation and conformation of the substrate disulfide bond. We
speculate that similar situations might happen in vivo without the need of reaching elevated
forces. For instance, it may be the case of DsbD, a member of the Trx superfamily present in
E. coli that interacts with cytoplasmic Trx37. DsbD is associated to the membrane and that
might restrict its mobility originating a situation in which the adaptability of E. coli Trx
might be essential for the transmembrane electron flow.
Although our experiments shed light onto the chemical evolution of Trx catalysis, by
examining extant organisms we can only see the end of the time line. An exhaustive tracking
of the chemical evolution of Trx catalysis would require the resurrection of ancient Trxs38.
This approach will allow us to more precisely determine the important chemical changes and
their relation to relevant biological phenomena that occurred in the distant past.
We anticipate that the enzymatic studies carried out on Trx at the single-molecule
level, can serve as starting point to investigate the chemistry of other enzymes such as C-S
lyases39 or proteases40 in which the catalyzed rupture of covalent bonds is the fundamental
process. Finally, we conclude that our findings represent a set of novel observations that
confirm force as innovative and sensitive tool capable of offering unprecedented details of
chemical reactions at the single-bond level.
Acknowledgments
We thank Dr. Sergi Garcia-Manyes for careful reading of the manuscript and all the
Fernandez laboratory members for helpful discussions. This work was supported by
National Institutes of Health Grants HL66030 and HL61228 to J. M.F
9
Material and Methods
Protein expression and purification
Preparation of (I27G32S-A75C)8 polyprotein has been extensively described in
previous works where the reader is referred for further details9,18. The expression and
purification of the different Trxs used have been also described elsewhere: P.
falciparum41, drosophila Trx142, poplar Trxh143 and Trxh344, Pea Trxm45, E. coli Trx146,
E. coli Trx213 and human Trx210.
Sequence analysis
Sequence alignment was carried out using ClustalW and modified by hand. Tree
topology and branch lengths of the tree were estimated using Mr. Bayes (v. 3.5) with rate
variation modeled according to a gamma distribution. The following GI numbers were
accessed from GenBank: Bacteria E. coli Trx1 (67005950), Salmonella Trx1
(16767191), E. coli Trx2 (16130507), Salmonella Trx2 (16765969), Human Trx2
mitochondria (21361403), Bovine Trx2 mitochondria (108935910), Rickettsia
(15603883), Nostoc (17227548), Proclorococcus (126696505), Spinach Trxm chloroplast
(2507458), Pea Trxm chloroplast (1351239), Thermus (46199687), Deinococcus
(15805968), Archaea Aeropyrum (118431868, Hyperthermus (124027987), Sulfolobus
(15897303), Eukaryote Plasmodium falciparum (75024181), Poplar Trx h1 (19851972),
Poplar Trx h3 (2398305), Pea (27466894), Dictyostelium (165988451), Bovine
(27806783), Human Trx1 (135773).
Single molecule force-clamp experiments
The details of our custom-made atomic force microscope have been described
previously47. We used silicon nitride cantilever (Veeco) with a typical spring constant of
20 pN/nm and were calibrated using the equipartition theorem. The force-clamp mode
provides an extension resolution of ~0.5 nm and a piezoelectric actuator feedback of
~5ms. The buffer used in all the experiments was: 10 mM HEPES, 150 mM NaCl, 1mM
EDTA, 2mM NADPH at pH 7.2. Before the beginning of the experiment, Trx reductase,
bacterial or eukaryotic depending on the case, was added to a final concentration of 50
nM. The different Trxs were added to the desired concentration. The reaction mixture
and the substrate were added and allowed to absorb onto a freshly evaporated goldcoverslip before the experiments. The force-clamp experiment consists of a double-pulse
force protocol. The first pulse was set at 175 pN during 0.3-0.4 s. The second pulse can
be set at different forces and was held time enough to capture all the possible reduction
events. The experiments using metallic Zn were carried out in citrate buffer 100 mM at
pH 6. After adding Zn nanoparticles (Sigma) to a concentration of 10 mM, the solution
was sonicated to allow resuspension. Due to the experimental difficulty of working at low
forces with Zn nanoparticles, we only include experiments at forces over 200 pN. The pH
of the buffer was verified during the time of the experiment and no significant changes
were observed. In addition, to verify the behavior of the substrate in citrate buffer, a
control experiment in the absence of Zn nanoparticles was carried out and no reduction
10
events were detected. Data collection and analysis were carried out using custom written
software in IGOR Pro 6.03 (Wavemetrics). The collected traces (15-50 per force)
containing the reductions events, were summated and averaged. The resulting averaged
traces were fitted with single exponential from where the rate constant can be obtained.
The analysis of the force-dependent reduction kinetics was carried out using the kinetic
model described in the next section.
11
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α0 (μM-1∙s-1)
β0 (s-1)
γ0 (μM-1∙s-1)
k10 (s-1)
Δx12 (Å)
Δx02 (Å)
E. coli Trx
0.24±0.02
29±2
0.017±0.003
4.4±0.6
-0.82±0.07
0.16±0.02
E. coli Trx2
0.19±0.02
27±2
0.013±0.005
5.0±0.6
-1.65±0.03
0.16±0.01
Human Trx2
0.60±0.09
29±2
0.021±0.003
4.4±0.5
-0.82±0.05
0.17±0.03
Pea Trxm
0.24±0.03
27±6
0.018±0.008
4.0±0.8
-0.97±0.09
0.16±0.02
P. falciparum Trx
0.37±0.07
26±2
0.054±0.012
24.8±0.5
-0.99±0.09
-
Human Trx1‫٭‬
0.43±0.09
35±6
0.15±0.04
2.9±0.7
-0.79
-
Poplar Trxh3
0.12±0.03
31±4
0.019±0.005
4.6±0.7
-1.32±0.08
-
Poplar Trxh1
0.19±0.02
28±2
0.028±0.009
4.6±0.6
-0.73±0.06
-
λ0(s-1)
Table 1. Kinetic parameters for Trxs from different species. The parameter where
obtained using the kinetic model described in supplementary information. They are the
result of numeric optimization of the global fit using the downhill simplex method. The
errors bars correspond to the standard error. * These values have been obtained from ref
9
.
15
Figure Legends
Fig 1. Phylogeny of Trx homologs from representative species of the three domains
of life. Branch lengths were estimated using maximum likelihood with rate variation
modeled according to a gamma distribution. Scale bar represents amino acid
replacements per site per unit evolutionary time. Posterior probabilities are shown at
nodes of the phylogeny when greater than 50%. The lack of strong node supports deep in
the phylogeny results from the ambiguous placement of mitochondrial sequences,
possibility due to long-branch attraction effects with non-bacterial sequences. In contrast,
there is strong support for the grouping of chloroplast and cyanobacteria (not shown).
Boxes highlight the proteins experimentally studied in this work.
Fig 2. Force-clamp experiments of single-disulfide reductions. (A) Graphic
representation of the force-clamp experiment for single-disulfide reductions by Trx. A
first force pulse rapidly unfolds the I27 modules exposing the buried disulfide bonds to
the solvent. The second force pulse is set to monitor single-disulfide reductions that are
uniquely identified by the extension of the residues trapped behind the disulfide bond.
(B) Example of trace showing the unfolding and consequent disulfide reductions of a
(I27G32S-A75C)8 polyprotein. In the example shown the unfolding pulse was set at 175 pN
for 0.3 s and the reduction pulse was set to 75 pN for several seconds. (C) Summed and
averaged traces of disulfide bond reductions at different forces for pea Trxm 10 µM and
(D) for poplar Trxh1 10 µM. The colored lines represent single-exponential fits from
where we can obtain the rate of reduction (r = 1/τ) at a given force.
Fig 3. Force-dependent chemical kinetics of disulfide reduction by Trxs from
different species. (A) Bacterial-origin Trxs: Human mitochondrial Trx2 (blue), E. coli
Trx1 (red), pea chloroplastic Trxm (black), E. coli Trx2 (green). While the MichaelisMenten mechanism differs in magnitude among the Trxs, the SN2 reaction at elevated
forces is identical and characteristic for all of them. The inset shows the expansion in the
force range where the reduction rate reaches the minimum value in each case. (B)
Eukaryotic-origin Trxs: Human Trx1 (blue, obtained from ref 9), Plasmodium (red),
Poplar Trxh1 (black), Poplar Trxh3 (green). The absence of the SN2 reaction at high
forces in all eukaryotic Trxs reveals a force-independent mode of disulfide reduction.
Similarly to bacterial Trxs, the insect shows the minimum rates of reduction. In all
experiments the concentration of Trx was 10 µM. The lines represent the fit using the
kinetic model described in the methods section. The kinetics parameters obtained are
summarized in Table 1.
Fig. 4. The three mechanisms of disulfide reduction detected by force-clamp
spectroscopy. A) reduction kinetics by different reducing agents: Human Trx (black
squares, from ref 9), L-cysteine (gray circles, from ref 20) and metallic Zn (red circles).
The disulfide reduction by metallic Zn proceeds through a force-independent reaction and
shows a striking similarity to that of eukaryotic Trxs at elevated forces. B) Schematic
representation of the Michaelis-Menten reduction mechanisms present in all Trxs. This
mechanism entails a geometrical reorientation of the substrate disulfide bond in order to
achieve the proper alignment for and SN2 reaction to occur. This causes the shortening of
16
the substrate polypeptide (-Δx). C) Representation of the transition state of the SN2
mechanism carried by bacterial Trxs when elevated forces are applied to the substrate.
This conformation is favored by a shallow binding groove allowing for the simple SN2
type lengthening of the substrate disulfide bond at the transition state, which is the origin
of the exponential dependency of the rate of reduction. D) Representation of the singleelectron transfer mechanism ubiquitous to all Trxs. This mechanism is more visible in
eukaryotic Trxs at high forces.
Fig.5. Computational structural analysis and molecular dynamics simulations. (A)
Geometrical dissection of the hydrophobic binding groove, shaded in green and outlined
in red for human Trx (pdb: 3trx). The depth and width of the groove in the region
surrounding the active cysteine are indicated by arrows and the active cysteine is colored
in orange. (B) Groove depth for three eukaryotic-origin Trxs (red): Human Trx
(pdb:1mdi); A. Thaliana Trxh1 (pdb:1xfl) and Spinach Trxf (pdb:1f9m), and three
bacterial-origin Trxs (blue): Human Trx2 (pdb:1uvz); E. coli Trx1 (pdb:2trx) and C.
reinhardtii Trxm (pdb:1dby). It is clear that the eukaryotic binding grooves are deeper
than their bacterial counterparts. (C) Molecular dynamics simulation of the substrate
mobility within the binding groove for different eukaryotic (red) and bacterial (blue)
Trxs. Three eukaryotic complexes were used: Human Trx with the substrate REF-1
(pdb:1cqg); Human Trx with NF-κB (pdb:1mdi) and barley Trxh2 with protein BASI
(pdb:2iwt). The two bacterial complexes were: E. coli Trx with Trx reductase,
(pdb:1f6m) and B. subtilis Trx bound to Arsc (pdb:2ipa). The large RMSD of the
bacterial Trxs (blue) indicate a high substrate mobility that may facilitate collisions
orientated so as favoring SN2 reactions. Eukaryotic Trxs (red) are highly restricted which
may explain the different chemical behavior at high forces as compared with bacterial
Trxs (Fig.4). These steric restrictions in eukaryotic Trxs could favor an SET mechanism
over SN2. (D) Diatomic distance distribution of the S-S bond in the Trx-substrate mixed
intermediate, using the same structures as in (C). Again we infer higher mobility of the
substrate in bacterial-origin Trxs as indicated by the broader distance distributions of the
bacterial complexes (blue) as compared to those of eukaryotic Trxs (red).
17
Fig.1
18
Fig. 2
19
Fig. 3
20
Fig. 4
21
Fig. 5
22