Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity

Abstract

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c(7) from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e(-)/H(+) transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e(-)/H(+) coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.

Linking Spectral and Electrochemical Analysis to Monitor c‐type Cytochrome Redox Status in Living Geobacter sulfurreducens Biofilms

“…[14,40] Its macroscopic redox potential is centered at À146 mV, although a cycle of protonation and deprotonation alters the microscopic redox potential of heme III to higher values (À123 mV) during oxidiation, and lower values (À185 mV) during reduction. [41,42] This potential window is consistent with the sharp transition seen in biofilm oxidation state at higher potentials, in the À150 mV range.…”

Transcriptomic and Genetic Analysis of Direct Interspecies Electron Transfer

“…2), suggesting a lack of selection for increased expression during DIET. Only PpcA and PpcD can accept and donate protons along with electrons (54). The expression level of the remaining three homologs of ppcA in G. sulfurreducens (ppcB, ppcC, and ppcE) was low in both types of cocultures (see Table S1e).…”

Harnessing the power of microbial nanowires

“…The periplasmic c‐Cyts PpcA and PpcD are particularly abundant in the cell envelope and are conserved among Geobacter species (Butler et al ., 2010). These c‐Cyts are predicted to contribute to the proton motive force by coupling the transport of electrons to protons, although using different mechanisms (Pessanha et al ., 2006; Morgado et al ., 2010). Energy transduction by these c‐Cyts can be sustained over a wide range of redox potentials (mid‐point potential versus the standard hydrogen electrode ranges from −0.167 to −0.109 V in PpcA and from −0.202 to −0.146 V in PpcD), allowing them to function as intermediate electron carriers to more than one redox partner (Morgado et al ., 2010).…”

“…These c‐Cyts are predicted to contribute to the proton motive force by coupling the transport of electrons to protons, although using different mechanisms (Pessanha et al ., 2006; Morgado et al ., 2010). Energy transduction by these c‐Cyts can be sustained over a wide range of redox potentials (mid‐point potential versus the standard hydrogen electrode ranges from −0.167 to −0.109 V in PpcA and from −0.202 to −0.146 V in PpcD), allowing them to function as intermediate electron carriers to more than one redox partner (Morgado et al ., 2010). The exposure of the base of the T4P fibres in the periplasm offers opportunities for interactions with the periplasmic electron carriers and electron discharges via the T4P pathway (Fig.…”