Thermodynamic and functional characterization of the periplasmic triheme cytochrome PpcA from Geobacter metallireducens

Abstract

The bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular electron acceptors, including pollutants or electrode surfaces for current production in microbial fuel cells. For these reasons, are of interest for practical biotechnological applications. The use of such electron acceptors relies on a mechanism that permits electrons to be transferred to the cell exterior. The cytochrome PpcA from is a member of a family composed of five periplasmic triheme cytochromes, which are important to bridge the electron transfer from the cytoplasmic donors to the extracellular acceptors. Using NMR and visible spectroscopic techniques, a detailed thermodynamic characterization of PpcA was obtained, including the determination of the heme reduction potentials and their redox and redox-Bohr interactions. These parameters revealed unique features for PpcA from compared with other triheme cytochromes from different microorganisms, namely the less negative heme reduction potentials and concomitant functional working potential ranges. It was also shown that the order of oxidation of the hemes is pH-independent, but the protein is designed to couple e/H transfer exclusively at physiological pH.

A unique aromatic residue modulates the redox range of a periplasmic multiheme cytochrome from Geobacter metallireducens

“…Compared to the wild-type cytochrome, the macroscopic redox behavior of the PpcAW45M was the most distinct and shifted by 30% the working redox potential range of the protein toward the one observed for PpcA from G. sulfurreducens (Table 1). For this reason, this mutant was selected for a detailed thermodynamic characterization in experimental conditions that match those previously reported for the wild-type protein (24). Therefore, the same set of heme methyl groups (2 1 CH 3 I , 12 1 CH 3 III and 2 1 CH 3 IV ) was used to probe the individual heme oxidation profiles in 2D 1 H, 1 H-EXchange SpectroscopY (EXSY) NMR spectra acquired in the pH range of 6.1-8.5.…”

“…The samples were fully reduced following the procedure indicated previously and were partially oxidized by first removing the hydrogen from the sample with argon and then by introducing controlled amounts of air with a Hamilton syringe. The NMR redox titrations for observation of the stepwise oxidation of the protein followed the same procedure described for the wild-type protein (24). The oxidation patterns of the same set of heme methyls used for the wildtype cytochrome were monitored by 2D 1 H, 1 H-EXSY spectra at 15 °C.…”

“…The assignment of the NMR signals was obtained as described for the wild-type protein (23,24). 1 H chemical shifts were referenced to DSS at 0 ppm and 15 N chemical shifts were calibrated through indirect referencing (28).…”

“…A preliminary structural study has suggested that these two proteins share a high structural homology with some differences near hemes I and III (23). However, a detailed functional characterization has shown that these two proteins possess markedly different mechanistic functional properties (24). In fact, the order of oxidation of the heme groups is different (IV-I-III in PpcA from G. metallireducens and I-IV-III in PpcA from G. sulfurreducens) as well as their apparent midpoint redox potential values (-93 mV for PpcA from G. metallireducens and -138 mV for PpcA from G. sulfurreducens, at pH 8).…”

Determining incremental coulombic efficiency and physiological parameters of early stage Geobacter spp. enrichment biofilms

“…Moreover, few members of this genus form multilayered biofilms at anodes and produce current densities of up to 0.8-1.0 mA cm −2 [19,20]. Therefore, Geobacter species are used as model organisms for studying DEET in terms of electron transfer kinetics [21], biochemical properties of redox centers [22,23], thermodynamics (including analyses of Gibbs free energy and enthalpy) [24,25], and biofilm conductivity [26,27].…”