Resonance Raman, EPR and MCD Spectroscopic Investigation of Diheme Cytochrome c Peroxidases from Nitrosomonas europaea and Shewanella oneidensis

Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one class of enzymes is active in the diferric form (constitutively active), and the other class of enzymes requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). In order to better understand the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( Ne CcP) and a reductively activated bCcP from Shewanella oneidensis ( So CcP) were characterized in both the diferric and semi-reduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in Ne CcP or So CcP in either the diferric or semi-reduced states in solution. This points towards a mechanism of activation where the active site L-heme is not in a static, five-coordinate state, but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme, and that forms a more stable Fe IV =O intermediate. Spectroscopic methods are used to interrogate the diferric and semi-reduced forms of bacterial Cyt. c peroxidases from Nitrosomonas europaea ( Ne CcP) and Shewanella oneidensis ( So CcP). Our results indicate that the L-heme is not in a static, five-coordinate state in the catalytically active forms of these enzymes, but that the activation mechanism of these enzymes is more subtle, and different from MauG.