Supplementary methods
Appendix A: Cloning of cytochrome PccH gene
The gene pccH was amplified with primers containing restriction sites for the enzymes
EcoRI
(5’-CCTTTGAATTCTCAGTACGTGACCGTGATGG-3’)
CTATTCTTGCGGCCGCTATGGGCCTTTTG-3’).
Fragment
size
and
NotI
(5’-
(456bp)
was
confirmed by 0.8% agarose gel electrophoresis. Ligation was carried out with an
insert:vector molar ratio of 5:1, for 16 hours at 16 °C. The ligation product was
transformed into E. coli DH5α competent cells prepared by the calcium chloride method
[1] and plated for selection in LB medium supplemented with ampicillin (100 µg/mL).
The resulting colonies were screened by colony PCR with pCK32 primers (pCK32_fw:
5’-GGCTCGTATGTTGTGTGGAA-3’
and
pCK32_rv:
5’-
AAGGGAAGAAAGCGAAAGGA-3’), and those with a PCR product of the correct
size were grown in liquid LB medium supplemented with ampicillin for plasmid
extraction and sequencing.
Production and purification of recombinant proteins
Escherichia coli strain BL21(DE3) (Novagen) containing the plasmid pEC86, which
codes for the c-type cytochrome maturation (ccm) operon that allows E. coli to produce
periplasmic cytochromes in aerobic conditions [2], was co-transformed with plasmid
pCS3274. Transformed E. coli cells were grown in 2xYT medium containing 34 µg/mL
chloramphenicol (Sigma) and 100 µg/mL ampicillin (Sigma). An isolated colony was
transferred from agar plates to 50 mL of 2xYT medium and grown overnight at 30°C.
The resulting cell culture was used for a 1% inoculum of two 2-liter conical flasks with
1 liter of 2xYT each, and the same growth conditions were used. After reaching a
OD600nm ~1.5, protein expression was induced with 100 µM isopropyl β-Dthiogalactopyranoside (IPTG) after overnight incubation. Cells were harvested by
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centrifugation, lysed by osmotic shock in presence of lysozyme and the periplasmic
fraction obtained as previously described for the Gs PpcA triheme cytochrome family
[3, 4].
The periplasmic fraction was concentrated in an ultrafiltration cell with a 3 kDa cut-off
membrane, and dialyzed twice against 10 mM Tris-HCl (pH 8). The resulting fraction
was loaded into two 5 mL Econo-Pac HighS cartridges (BioRad) connected together
and equilibrated with 10 mM Tris–HCl (pH 8), and eluted with a NaCl gradient from 0
to 300 mM in 10 mM Tris–HCl (pH 8). The fractions containing the cytochrome PccH
were pooled, concentrated, and the buffer was exchanged to 100 mM sodium potassium
phosphate buffer (pH 8) in an ultrafiltration cell with a 3-kDa cut-off membrane. The
resulting fraction was loaded into a Superdex 75 16/70 column (GE Healthcare) and
subjected to gel filtration chromatography. Both chromatography steps were performed
in an ÄKTA Prime Plus FPLC System (GE, Amersham). The presence of the
cytochrome
was
confirmed
by sodium
dodecyl
sulfate
polyacrilamide
gel
electrophoresis (SDS-PAGE) stained with Comassie Blue or by TMBZ/H2O2 to detect
hemes [5]. Production and purification of each PccH mutant was performed as
described for the wild-type protein.
Appendix B: Quantification and extinction coefficient of PccH
The concentration of purified cytochrome PccH was determined with the Pierce BCA
Protein Assay Kit (Thermo Scientific) using horse-heart cytochrome c as standard. UVvisible spectra were recorded in the range 350-700 nm, at room temperature for both
oxidized and reduced samples and used to determine the molar extinction coefficient of
cytochrome PccH.
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Appendix C: Molecular mass determination and heme quantification
The molecular mass of cytochrome PccH was determined by matrix-assisted laser
desorption−ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using a
Voyager-DETM PRO Biospectrometry workstation equipped with a nitrogen laser
radiating at 337 nm from Applied Biosystems (Foster City, United States). A matrix
solution of sinapinic acid in 70:30 water/acetonitrile with 0.1% TFA (final
concentration) was used. The measurements were performed in triplicates in positive
ion mode using ProteoMassTM cytochrome c MALDI-MS from Sigma-Aldrich (MW
12361.96 Da) as internal calibration.
The quantification of the c-type hemes present in PccH was obtained by the pyridine
hemochrome method, as described by Berry and Trumpower [6]. Purified protein (5 μM
final concentration) was initially incubated with 50 mM NaOH and 20% v/v pyridine in
aqueous solution at room temperature for 15 min. Two separated aliquots of the
resulting solution were then prepared by adding the reductant sodium dithionite
(pyridine ferrohemochrome form) and the oxidant potassium ferricyanide (pyridine
ferrihemochrome form). UV-visible spectra of both pyridine hemochrome forms were
recorded between 350-700 nm and the heme content was determined using the
difference absorption coefficient of 21.84 mM-1cm-1 at 550 nm for the pyridine
ferrohemochrome and the pyridine ferrihemochrome spectrum [6].
Appendix D: Redox titrations followed by visible spectroscopy and determination
of reduction potentials
To check for hysteresis, each redox titration was performed in both reductive and
oxidative directions, using sodium dithionite and potassium ferricyanide solutions,
respectively. To ensure a good equilibrium between the redox centre of the protein and
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the working electrode, a mixture of the following redox mediators was selected
according to the procedures defined in the literature [7] and added to the solution, all at
approximately 1.5 μM final concentration: p-benzoquinone, tetramethyl 1,4phenylenediamine,
1,2-naphtoquinone-4-sulfonic
acid,
1,2-naphtoquinone,
trimethylhydroquinone, phenazine methosulphate, phenazine ethosulphate, methylene
blue, indigo tetrasulphonate, indigo trisulphonate, and indigo disulphonate. These
mediators cover the potential range of 280 to -120 mV (relative to standard hydrogen
electrode, SHE). A combined Pt/Ag/AgCl electrode was used for measuring the solution
potential. The redox potential of the solution was measured after each addition of
reductant or oxidant agent, and once stable, a visible spectrum was recorded. The
reduced fraction of the protein was determined by integrating the area of the α-peak
(552 nm) above the line connecting the flanking isosbestic points (545 and 559 nm) to
subtract the optical contribution of the redox mediators, as described previously [8]. The
measured solution potentials were corrected to standard hydrogen electrode by adding
207 mV. The experiments were performed at least twice at each pH value, and the
reduction potentials were found to be reproducible within +/− 5 mV.
References
1.
Sambrook J, Russell DW (2001) Molecular Cloning: a laboratory manual. New
York, Cold Spring Harbor Laboratory Press.
2.
Arslan E, Schulz H, Zufferey R, et al. (1998) Overproduction of the
Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in
Escherichia coli. Biochem Biophys Res Commun 251, 744-747.
3.
Fernandes AP, Couto I, Morgado L, et al. (2008) Isotopic labeling of c-type
multiheme cytochromes overexpressed in E. coli. Prot Expr Purif 59, 182-188.
4.
Pokkuluri PR, Londer YY, Yang X, et al. (2010) Structural characterization of a
family of cytochromes c7 involved in Fe(III) respiration by Geobacter
sulfurreducens. Biochim Biophys Acta 1797, 222-232.
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5.
Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the
detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl
sulfate polyacrylamide gels. Anal Biochem 75, 168-176.
6.
Berry EA, Trumpower BL (1987) Simultaneous determination of hemes a, b,
and c from pyridine hemochrome spectra. Anal Biochem 161, 1-15.
7.
Dutton PL (1978) Redox potentiometry: determination of midpoint potentials of
oxidation-reduction components of biological electron-transfer systems.
Methods Enzymol 54, 411-435.
8.
Morgado L, Bruix M, Orshonsky V, et al. (2008) Structural insights into the
modulation of the redox properties of two Geobacter sulfurreducens
homologous triheme cytochromes. Biochim Biophys Acta 1777, 1157-1165.
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Supplementary figures
Figure S1 – Portion of a 2D 1H EXSY NMR spectrum of cytochrome PccH (25°C and
pH 7) obtained for a partially oxidized sample. Cross peaks connecting the signals of
the axial methionine CH3 protons in the reduced and oxidized forms are indicated. The
correspondent regions in the 1D 1H NMR spectra of fully reduced (PccHred), fully
oxidized (PccHox) and partial oxidized (PccHint) PccH samples are indicated.
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Figure S2 - 2D 1H 13C HSQC NMR spectrum of cytochrome PccH in the oxidized state
(25°C and pH 7). Labels relate with the heme methyl signals according to IUPAC-IUB
nomenclature [1] and with CH and CH2 signals of heme axial histidine (H21). The
peaks of the protons connected to the same carbon atom (CH2 groups) are linked by a
straight line.
S7
Figure S3 – (A) pH dependence of the reduction potential (Eh) of cytochrome PccH.
Data points are represented by triangles. The solid line is the result of the fit to the
Nernst equation for the transfer of one electron coupled to one proton according to the
model and equation indicated in panel B [2]. EP (+57 mV) and ED (-36 mV) correspond
to the reduction potential values of cytochrome PccH in the protonated and
deprotonated forms, respectively. pKox (5.0) and pKred (6.6) are the pKa values of the
fully oxidized and fully reduced protein, respectively.
S8
Figure S4 – Expansions of the low-field region of 1D 1H NMR spectra obtained for
cytochrome PccH in the oxidized form at different pH values (25°C). The heme methyl
signals (21CH3, 71CH3, 121CH3 and 181CH3) are labeled. The pH value of the
experiment is indicated on the left of each spectrum.
References
1.
Moss GP (1988) Nomenclature of tetrapyrroles. Recommendations 1986
IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Eur J
Biochem 178, 277-328.
2.
Dutton PL (1978) Redox potentiometry: determination of midpoint potentials of
oxidation-reduction components of biological electron-transfer systems.
Methods Enzymol 54, 411-435.
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