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 (O-2(center dot-)). O-2(center dot-) 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 similar to 1.0 x 10(5) 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 similar to 1 x 10(6) M-1 s(-1) to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of similar to 1.0 x 10(5) M-1 s(-1), fully consistent with the direct method. Proteomic analysis indicated that ONOO-, but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO- with Mn-III to yield Mn-IV and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO- and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.