Outer membrane cytochrome c, OmcF, from Geobacter sulfurreducens: High structural similarity to an algal cytochrome c6


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Putative outer membrane c-type cytochromes have been implicated in metal ion reducing properties of Geobacter sulfurreducens.1 OmcF (GSU2432), OmcB (GSU2731), and OmcC (GSU2737) are three such proteins that have predicted lipid anchors. OmcF is a monoheme cytochrome, whereas OmcB and OmcC are multiheme cytochromes. Deletion of OmcF was reported to affect the expression of OmcB and OmcC in G. sulfurreducens.1 The OmcF deficient strain was impaired in its ability to both reduce and grow on Fe(III) citrate probably because the expression of OmcB, which is crucial for iron reduction, is low in this strain. U(VI) reduction activity of this bacterium is also lower on deletion of OmcB or OmcF. The U(VI) reduction activity is affected more by the deletion of OmcF than by the deletion of OmcB.2

The soluble part of OmcF (residues 20–104, referred to as OmcFS hereafter) has sequence similarity to soluble cytochromes c6 of photosynthetic algae and cyanobacteria.1 The cytochrome c6 proteins in algae and cyanobacteria are electron transport proteins that mediate the transfer of electrons from cytochrome b6f to photosystem I and have high reduction potentials of about +350 mV and low pI.3, 4 The structures of seven cytochromes c6 have been previously determined.4–10 Further, a c6-like cytochrome (PetJ2) of unknown function was recently identified in Synechoccus sp. PCC 7002 with a reduction potential of +148 mV and high pI.11 Here, we report the structure of OmcFS and its remarkable structural similarity to that of cytochrome c6 from the green alga, Monoraphidium braunii. To our knowledge, OmcFS is the first example of a cytochrome c6-like structure from a nonphotosynthetic organism.


Cloning, expression, and purification of OmcF

The DNA fragment coding for the predicted soluble segment of cytochrome OmcF, from residue 20 to 104 (OmcFS), was amplified from the G. sulfurreducens genomic DNA and cloned into vector pLBM4 following the procedure for ligation-independent cloning previously described.12 The protein was expressed in E. coli strain BL21(DE3) cotransformed with plasmid pEC86 that harbored the cytochrome c maturation genes.13 After the cultures were grown to midexponential phase at 30°C at a shaking speed of 250 rpm they were induced with 10–20 μM IPTG. The shaker speed was then lowered to 200 rpm and incubation continued for 16–18 h (overnight) at the same temperature. Cells were harvested and the pellets were resuspended in TES buffer (100 mM Tris-HCl, pH 8.0, 20% sucrose, 0.5 mM EDTA) containing 0.5 mg/mL lysozyme and protease inhibitor cocktail for bacterial cells (Roche Diagnostics) and incubated at room temperature for 15 min. Thirty milliliters of the buffer were used to resuspend a pellet from 1 L of culture. Then an equal volume of ice-cold deionized water was added and the cells were incubated on ice with gentle shaking for 15 min and centrifuged at 12,000g for 30 min at 4°C. The supernatant was dialyzed against 20 mM sodium acetate, pH 5.0 and loaded onto 2 × 5 mL Econo-Pac High S cartridges (Bio-Rad) equilibrated with the same buffer. Protein was eluted with 250 mL of 20 mM sodium acetate, pH 5.0 that contained a gradient of 0–1M NaCl. Fractions containing OmcFS were combined, concentrated in Centricon YM3 units (Millipore), and loaded onto HiLoad 16/60 Superdex 75 gel filtration column (Amersham) equilibrated with 20 mM sodium acetate, pH 5.0 containing 100 mM NaCl and was eluted with the same buffer. The protein was eluted as a monomer. The expected MW is 9284 Da including the heme; the observed MW using electrospray mass spectrometry was 9275 Da. The yield of purified OmcFS is 7.5 mg from 1 L of cell culture.

Determination of redox potential

Anaerobic redox titrations followed by visible spectroscopy were performed with approximately 0.6 mg/mL protein solutions in 32 mM phosphate buffer (pH 7 and 8) with NaCl (100 mM final ionic strength) at 298 K, as described previously,14 using the following mixture of redox mediators: potassium ferricyanide, p-benzoquinone, tetramethyl 1,4-phenylenediamine, 1,2-naphtoquinone-4-sulfonic acid, 1,2-naphtoquinone, trimethylhydroquinone, phenazine methosulphate, phenazine ethosulphate, methylene blue, indigo tetrasulphonate, and indigo trisulphonate.

UV-visible spectroscopy

UV-visible spectra of OmcFS were measured on an Ultrospec 2100pro spectrophotometer. Protein sample at 0.1 mg/mL concentration was used. Reduction of the protein sample was achieved by adding an excess of sodium dithionite.


Initial screening to identify crystallization conditions of OmcFS was carried out with protein at 30 mg/mL in sodium phosphate buffer, pH 7.8 using the sitting-drop vapor-diffusion method at 298 K. The commercial crystallization screens Index, SaltRx, Crystal Screen, PEG-Ion (Hampton Research), and Wizard I & II (deCODE Genetics) were used. The screening was accomplished with 96-well Greiner plates using a Mosquito robot (TTP LabTech). Microcrystals were observed in the drop containing SaltRx screen-22 (1.2M tri-sodium citrate dihydrate, 0.1M Tris pH 8.5). Optimization attempted by variation of the ingredients of the original condition did not result in better sized crystals. The crystals were eventually optimized by a microseeding technique as follows: one crystal was transferred to 50 μL of reservoir solution (SaltRx-22) and was crushed; this constituted Seed-I solution. One microliter of Seed-I solution was diluted 50 times using reservoir solution generating Seed-II, which was further diluted 50 times resulting in the Seed-III solution. Protein at 53 mg/mL concentration (in buffer, 20 mM sodium acetate pH 5.0, 0.1M NaCl) was used for crystal optimization by the hanging-drop vapor-diffusion method in 24-well plates. One microliter of protein was mixed with 1 μL of reservoir solution (SaltRx-22) and 0.5 μL of Seed (I, II, or III) solution was added to the drop. The drops containing the seed solutions were allowed to equilibrate over 0.5 mL of reservoir solution. The best crystals were obtained using the Seed-II solution and were used for data collection.

Data collection and processing

Crystals were transferred to a cryoprotectant solution (SaltRx-22 containing 28% sucrose) for a few seconds and subsequently frozen by direct immersion in liquid nitrogen. Two diffraction data sets, one at 8 keV for structure solution using the Single wavelength Anomalous Dispersion (SAD) method and another at 12 keV for refinement, were collected at the Structural Biology Center 19BM beam line (Advanced Photon Source). The diffraction data were processed using the HKL3000 package.15 The crystallographic parameters and data collection statistics are shown in Table I.

Table I. Crystallographic Data and Refinement Statistics
  1. Values in parentheses refer to the highest resolution shell. Rfree is calculated using a random set of reflections (10%) not used in refinement.

Unit cell parameters  
a, b, c (Å)38.08, 40.19, 48.42 
Space groupP212121 
Molecules per asymmetric unit1 
VM3 Da−1)162.0 
  SAD data
Wavelength (Å)0.979111.54980
Resolution range (Å)50.0–1.30 (1.31–1.30)50.0–2.0 (2.07–2.00)
Rmerge (%)3.7 (10.5)3.4 (4.0)
Completeness (%)96 (70)94 (67)
Redundancy4.2 (2.7)7.6 (4.4)
Mean I/σ(I)42.4 (9.7)63 (31)
 FOM 0.49
 FOM (after density modification) 0.93
 Total no. of reflections33,541 
 σ – cutoff0.0 
 Rcryst (Rfree) (%)16.7 (18.3) 
 Wilson B-factor (Å2)5.9 
 No. of atoms [mean B-factor (Å2)]  
  Protein579 (8.6) 
  Heme43 (5.5) 
  Water117 (24.8) 
 r.m.s deviations from ideal geometry  
  Bond lengths (Å)0.006 
  Bond angles (°)2.1 

Structure determination and refinement

The structure of OmcFS was determined by the SAD method using the program CNS.17 The electron density map following density modification procedure using CNS was of excellent quality. Building of the model of OmcFS was accomplished manually using the programs Coot18 and CHAIN19 and refined using the program CNS. The structure of OmcFS, refined to 1.3 Å resolution, has 100% of the residues in the most favored regions of the Ramachandran plot with no residues in disallowed regions as calculated by the program Molprobity.20 The final refinement statistics are shown in Table I. The structure factors and atomic coordinates have been deposited in the Protein Data Bank (accession code: 3CU4).


Optical absorption spectrum

The UV-visible spectrum of purified OmcFS had peaks at 526, 410, and a shoulder at 364 nm. When reduced with sodium dithionite, the peaks were at 552.5, 522, and 416.5 nm.

Description of the structure

Electron density was observed from residue Gly26 at the N-terminus to Pro104 at the C-terminus. The electron density for residues 46 to 48 that are part of a loop region was weak. The protein has four helical segments (H1–H4) indicated in Figure 1(A,B). The heme axial ligands are His39 and Met79. The heme in OmcFS is surrounded by hydrophobic residues consisting of residues Phe31, Leu52, Ile62, Val68, Ile72, Phe82, Ile95, Tyr98, Val99, and Phe103. 117 water molecules were identified in the electron density. A very well-defined water molecule is located between the two propionic acid groups of the heme; this water molecule is also hydrogen bonded to hydroxyl group of Tyr71. A water molecule in a similar location has been observed in a number of cytochrome c6 structures.7

Figure 1.

A: Sequence alignment of OmcFS and Mb cyt. c6. The helical secondary structure as observed in OmcFS is indicated below the sequence. The acidic residues are boxed in red and the basic residues are boxed in blue. The residues boxed in gray are identical between the two proteins (in addition, note that the residues 58E in OmcF and 38E in Mb cyt. c6 are identical). B: Stereo view of the overlap of OmcFS and Mb cyt. c6 (PDB code, 1ctj) structures. Cα atoms of residues 7–37 and 73–80 in Mb cyt. c6 were overlapped with Cα atoms of residues 27–57 and 92–99 in OmcFS (RMS deviation 0.6 Å). Cα traces and the hemes are shown (black: OmcFS and gray: Mb cyt. c6); the numbers correspond to Cα positions in OmcFS.

Comparison with cytochromes c6 from green algae

OmcFS is a close structural homolog of the cytochrome c6 from green alga Monoraphidium braunii6 (Mb cyt. c6) [see Fig. 1(A,B)] with the DALI21 score of Z = 12.9. The sequence identity between the two proteins is 27%. The RMS deviation for the residues 27–60, 63–83, and 85–102 in OmcFS overlapped with residues 7–40, 45–65, 66–83 in Mb cyt. c6 is 1.1 Å. Five of the nine residues that form the hydrophobic core are identical between the two proteins. Interestingly, in the structure of Mb cyt. c6, Trp89 is equivalent to Phe103 of OmcFS in the hydrophobic core of the protein. Mb cyt. c6 has four extra residues at its C-terminus compared with OmcFS [Fig. 1(A)] and the chains have different conformations [Fig. 1(B)] resulting in the terminal residue Trp89 becoming part of the hydrophobic core.

The cytochrome c6 from Scenedesmus obliquus (So cyt. c6) is a close homolog of Mb cyt. c6. The structure of So cyt. c6 was determined in both the reduced and oxidized forms7 with the major difference observed between them was in the conformation of the Met residue that coordinates the heme. The torsion angles (χ1, χ2, χ3) of the methionine for the reduced and oxidized forms of So cyt. c6 are 160°, 65°, 178°, and 179°, 165°, 149°, respectively, resulting in a longer Fe to S distance in the reduced form. The chi values of the axial Met in OmcFS are 180°, 168°, 144° and that of Mb cyt. c6 are 178°, 164°, 153°. A comparison of the chi values of axial methionines suggests that the hemes are in the oxidized state in the structures of OmcFS and Mb cyt. c6.

Difference in reduction potentials of OmcFS and Mb cyt. c6

The reduction potentials of OmcFS were determined from the fit of the experimental data with a Nernst equation with n = 1, giving the values of +180 mV and +127 mV at pH 7 and 8, respectively. The redox titration curves are shown in Figure 2. The reduction potential of OmcFS shows significant pH dependence (redox-Bohr effect) in this pH range. A higher reduction potential is observed for the lower pH value, indicating the existence of positive heterotrophic cooperativity between the electrons and the protons, as expected on the basis of an electrostatic interaction between the heme iron and an acid/base group in its vicinity. Although the structures of OmcFS and Mb cyt. c6 are very similar their reduction potentials differ significantly: the reduction potential of the OmcFS is +180 mV at pH 7, whereas the reduction potential of Mb cyt. c6 is +358 mV at pH 7. Further, essentially no redox-Bohr effect was detected between pH 7 and 8 for Mb cyt. c6.22

Figure 2.

Redox titrations followed by visible spectroscopy for OmcFS at pH 8 (closed circles) and pH 7 (open circles). The solid lines indicate the results of the fits to the Nernst curves for one-electron reduction with +127 ± 5 mV (pH 8) and +180 ± 5 mV (pH 7). The reduction potentials are relative to the standard hydrogen electrode.

The heme is mostly buried in both OmcFS and Mb cyt. c6 structures with the heme exposed surface areas of 55 Å2 and 35 Å2, respectively, as calculated by the program Surface.23 The angles between the axial histidine ring plane and the heme porphyrin ring plane for OmcFS and Mb cyt. c6 are 84° and 88°, respectively. Although the two proteins have very similar structures the differences in sequence [see Fig. 1(A)] resulting in a different distribution of the polar and charged residues of the two proteins could contribute to the large difference in their redox potentials. The surface electrostatic potential for both OmcFS and Mb cyt. c6 as calculated by the program GRASP24 is shown in Figure 3. The differences in surface electrostatic potential clearly show that the two proteins will have different interacting partners in vivo. The Mb cyt. c6 is a more acidic protein; it has six more acidic residues and three fewer basic residues than OmcFS [Fig. 1(A)]. The calculated pI is 4.2 for Mb cyt. c6 and the measured pI is 3.6,22 whereas the calculated pI for OmcFS is 7.8 (pI values calculated with ExPASy ProtParam, without including the heme propionate groups in the calculation). There is a cluster of Arg residues (54, 57, and 63) near the heme in OmcFS, whereas only one basic residue (Lys34) is present in Mb cyt. c6. There is a cluster of four potentially negatively charged residues (Asp69, Glu70, Asp71, Glu72) on the surface of the protein on the side opposite from the heme-containing edge in Mb cyt. c6; only one acid group (Asp91) is present in equivalent position in OmcFS. Additionally in OmcFS, Lys50 is placed between the propionic acid groups of the heme forming a hydrogen bond with propionate A of the heme; His30 is the equivalent residue in Mb cyt. c6. Another residue worth noting is His47 in OmcFS. Although disordered in the crystal structure, the side chain of His47 is close to propionate D of the heme; the equivalent residue in Mb cyt. c6 is Ile27. The extra histidine close to the heme propionate groups might explain the significant redox-Bohr effect observed in the case of OmcFS.

Figure 3.

Surface electrostatic potential calculated by the program GRASP24: (A) OmcFS and (B) Mb cyt. c6. Red indicates negative and blue indicates positive potential. The scale of the potential ranges from −6 to 6 kT. The heme atoms are shown as green spheres with a propionic acid at the lower part of the figure. Two orientations separated by a rotation of about 180° are shown.

The amino acid sequences, structures, and redox potentials of cytochrome c6 molecules from photosynthetic organisms, algae and cyanobacteria, are very similar to each other as reviewed by Dikiy et al.10 and Worrall et al.4 Although the structure of OmcFS is similar to that of cytochrome c6 OmcFS is a more basic protein and has a much lower redox potential. In addition, although cytochrome c6 molecules are soluble proteins, OmcF is predicted to be attached to the membrane. The reduction potential and surface electrostatic potential of OmcF clearly show that its function is different from that of cytochrome c6 proteins from photosynthetic organisms.


The use of SBC beamlines and APS are supported by DOE under contract No. DE-AC02-06CH11357.