The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

The synthesis of [(η-C5H5)Mo(CO)3]2 satisfies many of the criteria for an exemplary experiment illustrating organometallic chemistry.

Understanding how a sequence of amino acids serves as a functional, catalytic unit is a fundamental question in enzymology. The Elliott group studies the structure‐function relationship using bacterial cytochrome c peroxidases (bCCPs) as a model system for probing the interplay between structure, redox chemistry, and catalysis. bCCPs are diheme periplasmic enzymes that reduce hydrogen peroxide to water, requiring two c‐type heme cofactors and two electrons from the cytochrome c pool. Peroxide detoxification is an essential bacterial defense mechanism. A hallmark of bCCPs lies in the potentials of their hemes: a low potential peroxidatic heme (FeL) and a high potential electron transfer heme (FeH). Interestingly, the bCCPs from Shewanella oneidensis (SoCCP) and Nitrosomonas europaea (NeCCP) feature a high sequence identity (60%), yet have key catalytic differences: SoCCP requires reductive activation of FeH, where NeCCP does not. Further, the reduction potentials governing respective FeH are very different: where SoCCP (+245 mV vs NHE) is average for bCCPs, and NeCCP (>400 mV vs NHE) is unusually high. Previous experiments in the Elliott group have found that point mutants in one heme environment can globally affect the redox properties. Specifically, mutating a key glutamate to lysine at FeL, which is expected to directly hinder activity, has unexpectedly altered the reduction potentials of FeH as well as FeL. The high sequence and structural similarities, yet differences in electrochemical properties, present a robust model to probe the influence of nearby residues on redox potentials and to understand bacterial defense mechanisms against damaging oxygen species.

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