Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34

Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2•–). O2•– reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO–). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 105 M–1 s–1 for the reaction of hMnSOD with ONOO– by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO–, including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO– at a rate of ∼1 × 106 M–1 s–1 to yield the fluorescence product, 7-hydroxycoumarin. Time-resol...