Rubrerythrin-catalyzed substrate oxidation by dioxygen and hydrogen peroxide

Investigations were undertaken aimed at distinguishing and clarifying the reactivities of the Fe(SCys)4 and diironoxo sites of rubrerythrin (Rr) during its catalysis of substrate oxidations by either dioxygen or hydrogen peroxide. Three Rr-catalyzed reactions were investigated: (1) the ferroxidase reaction: Feaq 2++O2→Feaq 3++[O]red; (2) the NADH peroxidase reaction: NADH+H++H2O2→NAD++2H2O; and (3) the aromatic diamine peroxidase reaction exemplified with o-dianisidine as substrate: o-dianisidine+H2O2→o-dianisidine quinonediimine+2H2O. A non-native bacterial oxidoreductase was used as a co-catalyst for the NADH peroxidase reaction. Residues at or near both metal sites of Rr, including those furnishing iron ligands, were mutated to assist in clarifying the metal-site reactivities. In addition a Rr with Zn2+ substituted for iron in the Fe(SCys)4 site was examined. The results indicate that, in reactions 1 and 2, electrons from the reductant flow initially into the Fe(SCys)4 site of Rr, then out through the diferrous site into O2 or H2O2. In reaction 3 oxidized Rr appears to weakly activate H2O2 for oxidation of the aromatic diamine substrate. The highest turnover occurs for the NADH peroxidase reaction. It is proposed that an extra carboxylate ligand not present in other diironoxo enzymes shifts the reactivity of the diferrous site of Rr towards hydrogen peroxide and away from dioxygen.