Outer membrane cytochrome <i>c</i>, OmcF, from <i>Geobacter sulfurreducens</i>: High structural similarity to an algal cytochrome <i>c</i>₆

Abstract

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. 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. 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