The detection of the nitrite reductase and NO-generating properties of haemoglobin by mitochondrial inhibition

Abstract

Nitrite (NO₂⁻), now regarded as an endocrine reserve of nitric oxide (NO), is bioactivated by nitrite reductase enzymes to mediate physiological responses. In blood, haemoglobin (Hb) catalyses nitrite reduction through a reaction modulated by haem redox potential and oxygen saturation, resulting in maximal NO production around the Hb P₅₀. Although physiological studies demonstrate that Hb-catalysed nitrite reduction mediates cyclic guanosine monophosphate (cGMP)-dependent vasodilation, the NO-scavenging effects of Hb raise questions about how NO generated from this reaction escapes the Hb molecule to signal at distant targets. Here, we characterize the NO-generating properties of Hb using the cGMP-independent and NO-dependent inhibition of mitochondrial cytochrome c oxidase. Using a novel technique to measure respiratory inhibition of isolated rat mitochondria, we provide evidence that the reduction of nitrite by intact red blood cells (RBCs) and Hb generates NO, which inhibits mitochondrial respiration. We show that allosteric modulators, which reduce the haem redox potential and stabilize the R state of Hb, regulate the ability of this reaction to inhibit respiration. Finally, we find that the rate of NO generation increases with the rate of Hb deoxygenation, explained by an increase in the proportion of partially deoxygenated R-state tetramers, which convert nitrite to NO more rapidly. These data reveal redox and allosteric mechanisms that control Hb-mediated nitrite reduction and regulation of mitochondrial function, and support a role for Hb-catalysed nitrite reduction in hypoxic vasodilation.