Nitric oxide dioxygenation in mammalian myoglobins and microbial flavohemoglobins

Abstract

All hemoglobins catalyze a reaction in which heme-bound dioxygen reacts directly with nitrogen monoxide (NO) to produce nitrate, resulting in the oxidation of the heme iron. This reaction, NO dioxygenation, has been proposed to proceed through a peroxynitrite intermediate that internally isomerizes to nitrate prior to release from the distal pocket of the globin. The work in this thesis examines the decay of the putative intermediate, using multiple myoglobin mutants to ascertain the role of key stereochemical features in the distal pocket. A reaction scheme is proposed that, we believe, provides a detailed mechanism for the formation and decay of a high-spin, cis-peroxynitrite-Fe(III) intermediate in the NO dioxygenation reaction. In mammalian myoglobins and hemoglobins the rate-limiting step for catalytic NO dioxygenase activity is the slow re-reduction of the heme by auxiliary enzymes, often flavin containing cytochrome b5 reductases. Flavohemoglobins are a family of microbial hemoglobins that contain a FAD-binding reductase domain attached directly to the globin, allowing for rapid re-reduction of the heme iron in the presence of NADH. As a result, these two-domain globins are extremely efficient NO dioxygenases that detoxify NO and are part of a microbial response to host defense mechanisms. Three fungal flavohemoglobins, one from Candida albicans and two from Aspergillus fumigates, have been isolated and preliminarily characterized with respect to ligand binding and NO dioxygenation activity. All three are functional flavohemoglobins as well as active NO dioxygenases. In contrast to other flavohemoglobins characterized thus far, AfFhbA exhibits several peculiar characteristics, the most striking being its unusual stability in the oxy complex. These results suggest the possibility of a different, as of yet undefined, function for this protein.