The
triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant under several growth conditions and is important
for extracellular electron transfer. PpcA plays a central role in
transferring electrons resulting from the cytoplasmic oxidation of
carbon compounds to the cell exterior. This cytochrome is designed
to couple electron and proton transfer at physiological pH, a process
achieved via the selection of dominant microstates during the redox
cycle of the protein, which are ultimately regulated by a well-established
order of oxidation of the heme groups. The three hemes are covered
only by a polypeptide chain of 71 residues and are located in the
small hydrophobic core of the protein. In this work, we used NMR and
X-ray crystallography to investigate the structural and functional
role of a conserved valine residue (V13) located within van der Waals
contact of hemes III and IV. The residue was replaced by alanine (V13A),
isoleucine (V13I), serine (V13S), and threonine (V13T) to probe the
effects of the side chain volume and polarity. All mutants were found
to be as equally thermally stable as the native protein. The V13A
and V13T mutants produced crystals and their structures were determined.
The side chain of the threonine residue introduced in V13T showed
two conformations, but otherwise the two structures did not show significant
changes from the native structure. Analysis of the redox behavior
of the four mutants showed that for the hydrophobic replacements (V13A
and V13I) the redox properties, and hence the order of oxidation of
the hemes, were unaffected in spite of the larger side chain, isoleucine,
showing two conformations with minor changes of the protein in the
heme core. On the other hand, the polar replacements (V13S and V13T)
showed the presence of two more distinctive conformations, and the
oxidation order of the hemes was altered. Overall, it is striking
that a single residue with proper size and polarity, V13, was naturally
selected to ensure a unique conformation of the protein and the order
of oxidation of the hemes, endowing the cytochrome PpcA with the optimal
functional properties necessary to ensure effectiveness in the extracellular
electron transfer respiratory pathways of G. sulfurreducens.
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.
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