Geobacter sulfurreducens Cytochrome c Peroxidases: Electrochemical Classification of Catalytic Mechanisms

Abstract

Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for “low reduction potential”) active site heme, and a secondary heme (H, for “high reduction potential”) associated with electron transfer, and an enzymatic activity that exists only when the H-heme is pre-reduced to the FeII oxidation state. The pre-reduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an “open” conformation allowing hydrogen peroxide to bind to the FeIII of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require pre-reduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool in distinguishing the electrocatalytic mechanisms of the Nitromonas-type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens, and the Geobacter S134P/V135K double mutant, which has been shown to be similar to the canonical sub-class of peroxidases and the Nitrosomonas sub-class of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH, yet is at a potential that is very similar to the H-heme. These findings are interpreted in terms of a model where rate-limiting intra-protein electron transfer governs the catalytic performance of the S134P/V135K enzyme.

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate

“…Electrochemistry on graphite electrodes has been previously reported for a number of bCcP family members, yet all previous studies used enzyme adsorbed in the as-isolated state. Direct electrochemistry of activatable bCcPs from Pseudomonas aeruginosa and Geobacter sulfurreducens , adsorbed with both hemes in the ferric oxidation state resulted in hydrogen peroxide turnover only in a low potential regime (∼ –100 mV vs SHE) that monitored an off-pathway Fe II /Fe III couple. In contrast, the constitutively active bCcP from Nitrosomonas europaea gave a single catalytic feature at high potential ( E cat > 500 mV) consistent with an on-pathway catalytic intermediate .…”

Electrocatalysis by Heme Enzymes—Applications in Biosensing

“…The electrochemical response of CcPs has been demonstrated at pyrolytic graphite (PG) and Au surfaces, using DET and MET [153][154][155][156]. In the latter case, physiological redox partners, including c-type cytochromes and small blue copper proteins, typically act as mediators [155][156][157][158].…”

Multi-heme proteins: Nature's electronic multi-purpose tool

“…Indeed the CcpA protomer adopts the same fold as other canonical bC c P enzymes, displays the identical requirement for reductive activation, and has sequence elements in Loops 1, 2 and 3 that suggest a strong similarity to Pa -type of bC c P enzyme. While mutations in Loop 1 what would render the sequence more like the Ne enzyme (and thereby presumably convert the enzyme to be constitutively active) did not perturb the reactivity of CcpA, the S134V/P135K double mutant in Loop 2 did (in part) achieve Ne enzyme-like activity [40, 42]. The resulting structure of the fully oxidized form of S134V/P135K shows a partially converted enzyme, where Loop 1 has shifted to an open conformation such that substrate might bind, yet Loop 2 is only partially reorganized as it should be in semi-reduced bC c Ps, and Loop 3 failed to convert to the required, semi-reduced conformation [40].…”

“…The resulting structure of the fully oxidized form of S134V/P135K shows a partially converted enzyme, where Loop 1 has shifted to an open conformation such that substrate might bind, yet Loop 2 is only partially reorganized as it should be in semi-reduced bC c Ps, and Loop 3 failed to convert to the required, semi-reduced conformation [40]. A further functional result of this partial inter-conversion between enzymatic conformers, was an apparent control of inter-cofactor electron transfer upon electrocatalytic reduction of substrate [42]. …”