Searching for the basis of constitutive activity in bacterial cytochrome c peroxidases

Bacterial cytochrome c peroxidases are marked by their possession of a c-type peroxidative active site in addition to a six-coordinate His/Met-ligated c-type heme associated with electron-transfer. The majority of the reported bCcPs are inactive when both hemes are in the ferric oxidation state (diferric). For these bCcPs, the peroxidatic heme environment is only ready for catalysis when the His/Met-ligated heme is first reduced to the ferrous oxidation state. In contrast, the bCcP family members from Nitrosomonas europaea (Ne) and Methylococcus capsulatus (Mc) have been reported to be active in the diferric state. Where Ne enzyme has proven to be a workhorse for structural, mutagenic and spectroscopic studies, the Mc enzyme has only been reported from the native organism and partially characterized. Here we report the recombinant expression of the Mc enzyme and reveal that it possesses reactivity that is in excellent agreement with the Ne system. Bioinformatics analyses do not reveal a shared primary sequence similarity between the Mc and Ne enzymes which might distinguish them from the activatable bCcPs. Phylogenetic analysis further supports how the Ne enzyme is found in clades that include the activatable enzymes. Together these data reveal that the Mc enzyme may be an additional useful platform to probe the reactivity of bCcP family members.