A Stable Ferryl Porphyrin at the Active Site of Y463M BthA.

BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)═O oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here, we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)═O porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O2 or H2O2. The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.

A widely distributed diheme enzyme from Burkholderia that displays an atypically stable bis-Fe(IV) state.

Bacterial diheme peroxidases represent a diverse enzyme family with functions that range from hydrogen peroxide (H2O2) reduction to post-translational modifications. By implementing a sequence similarity network (SSN) of the bCCP_MauG superfamily, we present the discovery of a unique diheme peroxidase BthA conserved in all Burkholderia. Using a combination of magnetic resonance, near-IR and Mössbauer spectroscopies and electrochemical methods, we report that BthA is capable of generating a bis-Fe(IV) species previously thought to be a unique feature of the diheme enzyme MauG. However, BthA is not MauG-like in that it catalytically converts H2O2 to water, and a 1.54-Å resolution crystal structure reveals striking differences between BthA and other superfamily members, including the essential residues for both bis-Fe(IV) formation and H2O2 turnover. Taken together, we find that BthA represents a previously undiscovered class of diheme enzymes, one that stabilizes a bis-Fe(IV) state and catalyzes H2O2 turnover in a mechanistically distinct manner.

Resonance Raman, Electron Paramagnetic Resonance, and Magnetic Circular Dichroism 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 is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced 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 NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which 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 FeIV═O intermediate.