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: Fe aq 2+ +O 2 →Fe aq 3+ +[O] red ; (2) the NADH peroxidase reaction: NADH+H + +H 2 O 2 →NAD + +2H 2 O; and (3) the aromatic diamine peroxidase reaction exemplified with o -dianisidine as substrate: o -dianisidine+H 2 O 2 → o -dianisidine quinonediimine+2H 2 O. 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 Zn 2+ 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 O 2 or H 2 O 2 . In reaction 3 oxidized Rr appears to weakly activate H 2 O 2 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.