Abstract The monoheme c-type cytochrome PccH from Geobacter sulfurreducens, involved in the pathway of current-consumption in biofilms, was electrochemically characterized in detail. Cyclic voltammetry was used to determine the kinetics and thermodynamics properties of PccH redox behavior. Entropy, enthalpy and Gibbs free energy changes associated with the redox center transition between the ferric and the ferrous state were determined, indicating an enhanced solvent exposure. The midpoint redox potential is considerably low for a monoheme c-type cytochrome and the heterogeneous electron transfer constant rate reflects a high efficiency of electron transfer process in PccH. The midpoint redox potential dependence on the pH (redox-Bohr effect) was investigated, over the range of 2.5 to 9.1, and is described by the protonation/deprotonation events of two distinct centers in the vicinity of the heme group with pKa values of 2.7 (pKox1); 4.1 (pKred1) and 5.9 (pKox2); 6.4 (pKred2). Based on the inspection of PccH structure, these centers were assigned to heme propionic acids \{P13\} and P17, respectively. The observed redox-Bohr effect indicates that PccH is able to thermodynamically couple electron and proton transfer in the G. sulfurreducens physiological pH range.

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2 Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15 Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c7-type domains (A, B, C and D) with homology to triheme cytochromes c7. In cytochromes c7 all three hemes are bis–His coordinated, whereas in c7-type domains the last heme is His–Met coordinated. The full-length GSU1996 has a 12 nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a “nanowire” of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c7-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c7-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c7-type domains from one multiheme protein were compared.

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III–I–IV for PpcB, as opposed to I–IV–III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.