Abstract-Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L -1 s -1 (pH 7.4, 37°C). Because the IC 50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 mol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling. (Circ Res. 2007;100:654-661.)

Nitrite (NO2 −) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II–IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

Nitrite represents a bioactive reservoir of nitric oxide (NO) that may modulate vasodilation, respiration and cytoprotection after ischemia-reperfusion injury. Although nitrite formation is thought to occur via reaction of NO with oxygen, this third-order reaction cannot compete kinetically with the reaction of NO with hemoglobin to form nitrate. Indeed, the formation of nitrite from NO in the blood is limited when plasma is substituted with physiological buffers, which suggests that plasma contains metal-based enzymatic pathways for nitrite synthesis. We therefore hypothesized that the multicopper oxidase, ceruloplasmin, could oxidize NO to NO+, with subsequent hydration to nitrite. Accordingly, plasma NO oxidase activity was decreased after ceruloplasmin immunodepletion, in ceruloplasmin knockout mice and in people with congenital aceruloplasminemia. Compared to controls, plasma nitrite concentrations were substantially reduced in ceruloplasmin knockout mice, which were more susceptible to liver infarction after ischemia and reperfusion. The extent of hepatocellular infarction normalized after nitrite repletion. These data suggest new functions for the multicopper oxidases in endocrine NO homeostasis and nitrite synthesis, and they support the hypothesis that physiological concentrations of nitrite contribute to hypoxic signaling and cytoprotection.