Rational design of electron/proton transfer mechanisms in the exoelectrogenic bacteria Geobacter sulfurreducens

Abstract

The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively produced family of five triheme cytochromes (PpcA−E) from the bacterium Geobacter sulfurreducens plays a crucial role in extracellular electron transfer, a hallmark that permits this bacterium to be explored for several biotechnological applications. Two members of this family (PpcA and PpcD) couple electron/proton transfer in the physiological pH range, a feature not shared with PpcB and PpcE. That ability is crucial for G. sulfurreducens’ growth in Fe(III)-reducing habitats since extra contributors to the electrochemical gradient are needed. It was postulated that the redox-Bohr effect is determined by the nature of residue 6, a leucine in PpcA/PpcD and a phenylalanine in PpcB/PpcE. To confirm this hypothesis, Phe6 was replaced by leucine in PpcB and PpcE. The functional properties of these mutants were investigated by NMR and UV-visible spectroscopy to assess their capability to couple electron/proton transfer in the physiological pH range. The results obtained showed that the mutants have an increased redox-Bohr effect and are now capable of coupling electron/proton transfer. This confirms the determinant role of the nature of residue 6 in the modulation of the redox-Bohr effect in this family of cytochromes, opening routes to engineer Geobacter cells with improved biomass production.

Cytochrome expression shifts in Geobacter sulfurreducens to maximize energy conservation in response to changes in redox conditions

“…The use of PpcB/PpcE could be an approach to decrease ATP production when the energy gradient is limiting, while PpcA is used when more energy is available. Thus, our results are consistent with the hypotheses that PpcA/PpcD are associated with ATP production (Morgado et al 2010; Silva, Portela, and Salgueiro 2021). Analyses of these periplasmic cytochromes have also discussed differences in working potentials, which could indicate different electron acceptors (Morgado et al 2010).…”

“…Interestingly, further studies have confirmed that PpcA and PpcD perform a proton-coupled electron transfer; which leads to the possibility that these periplasmic cytochromes are associated to ATP production (Morgado et al 2010). It has also been confirmed that electron transfer by PpcB and PpcE are not proton coupled, suggesting a different cellular function compared to PpcA/PpcD (Silva, Portela, and Salgueiro 2021).…”

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE

“…7a, b ) could be more advantageous than binding to another region of the monomer because such a binding pocket would not exist for monomeric OmcS, thus making the interaction specific to the nanowire and limit non-specific interaction between PpcA-E and OmcS monomers. Building on our studies to couple electron and proton transfer in PpcA-E via their rational design 14 , 54 , future studies to rationally engineer the interface between nanowires and PpcA-E could enable the control of electron injection rate by bacteria during EET and DIET. Such design approaches could enable control over EET and DIET rates to improve microbial performance.…”

“…The most affected residues were determined using amino acid-specific chemical shift mapping 42 (see Methods for details). Analysis of the PpcA backbone residues affected by the presence of OmcS (Δδ combined > Δδ cut-off ) shows two different extents of variation: residues Ile 4 , Ala 8 , Val 13 , Lys 14 , Phe 15 , Lys 18 , Ala 46 , His 47 , Gly 48 are the most affected ones by the presence of OmcS (Δδ combined > 1.5Δδ cut-off ), followed by Asp 3 , His 20 , Cys 30 , His 31 , Lys 37 , Lys 52 , Cys 54 , Thr 63 and Cys 68 (Δδ combined > Δδ cut-off ). The most affected residues comprise a region near heme IV (Ala 8 , Val 13 , Ala 46 , His 47 , Gly 48 ) and in between hemes I and III (Ile 4 , Lys 14 , Phe 15 , Lys 18 ) (Fig.…”