Functionally Distinct Bacterial Cytochrome <i>c</i> Peroxidases Proceed through a Common (Electro)catalytic Intermediate

The diheme cytochrome <i>c</i> peroxidase from <i>Shewanella oneidensis</i> (<i>So</i> 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 <i>So</i> 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 <i>c</i> peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome <i>c</i> peroxidase from <i>Nitrosomonas europaea</i> (<i>Ne</i>), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of <i>So</i> 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 <i>Ne</i> CcP. The pH dependence of catalysis for wild-type <i>So</i> CcP suggests that reduction shifts the p<i>K</i>_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 <i>Ne</i> 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