Myoglobin Is a Nitrite Reductase That Generates NO and Regulates Mitochondrial Respiration.

Abstract

Previous studies have demonstrated that nitrite is an endocrine store of NO that may play an important role in hypoxic vasodilation. Nitrite can be converted to NO enzymatically by deoxyhemoglobin, in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. In this study, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 50-times faster than deoxyhemoglobin due to its lower redox potential. Spectrophotometric measurements show the deoxymyoglobin-nitrite reaction to be second order and linearly dependent on deoxymyoglobin, nitrite and proton concentration with a bimolecular rate constant of 11.7 M−1s−1 at pH 7.4 at 37 degrees Celsius. Using this bimolecular rate constant, we calculate that at physiological concentrations of nitrite (20uM) and myoglobin (25uM), NO will be generated at a rate of 2.85 nM/sec. Since the IC50 of inhibition of mitochondrial respiration is approximately 100nM at physiological oxygen tensions (5–10 μM), we hypothesized that the myoglobin-dependent reduction of nitrite could regulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin in conjunction with nitrite to isolated rat liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in a measurable production of NO (with 1mM nitrite addition) and the inhibition of mitochondrial respiration (with 25μM nitrite addition) that was not significantly changed by the addition of the xanthine oxidase inhibitor allopurinol. These data corroborate previous studies demonstrating that NO generated from nitrite reduction can escape heme autocapture to mediate biological responses and confirm that the regulation of mitochondrial respiration by the nitrite-myoglobin reaction is relevant even in a physiological milieu. These data have implications for the modulation of hypoxic respiration, nitrite-dependent hypoxic signaling in tissue, and the regulation of oxygen gradients in the microcirculation.