Authors

Abstract

A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple electron and proton transfer through the redox-Bohr effect observed in the physiological pH range. In contrast, the capability of coupling electron and proton transfer was severely decreased in cytochromes PpcB and PpcE. The optimization of the redox-Bohr effect in microorganism involved in EET is conceivably a promising strategy to improve their performance. As part of understanding the molecular control of the redox-Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in both proteins PcpA and PpcD, which are capable of e-/H+ coupling, whereas in the other two characterized members incapable of such coupling (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox-Bohr center, we replaced Leu6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native, the mutant PpcAL6F is not able to couple e-/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replace Phe6 in these two proteins by leucine) and show that the mutant proteins showed an increased redox-Bohr effect. The results clearly establish the role of residue 6 in the control of the redox-Bohr effect in this family of cytochromes, a feature that can enable rational design of G. sulfurreducens strains carrying mutant cytochromes with optimal redox-Bohr effect as suitable for various biotechnological applications.