The chemistry of the nitrate-nitrite-nitric oxide pathway: regulating muscle oxygenation and exercise performance

Nitric oxide (NO) is a pleiotropic signaling molecule fundamentally involved in regulating skeletal muscle physiology, including blood flow, contractility, and metabolism. For decades, the synthesis of NO was attributed solely to the l-arginine-dependent nitric oxide synthase (NOS) enzymes. However, the discovery and characterization of the nitrate-nitrite-NO pathway have revealed an alternative, NOS-independent mechanism for NO generation. This pathway is particularly significant under hypoxic and acidic conditions, which are characteristic of exercising skeletal muscle. Dietary inorganic nitrate, abundant in green leafy vegetables and beetroot, is sequentially reduced to nitrite and then to bioactive NO. This review critically examines the intricate chemistry underpinning this pathway, from the initial enzymatic reduction of nitrate by both mammalian and microbial reductases to the diverse chemical routes of nitrite reduction to NO within the muscle milieu. We delve into the specific roles of key proteins such as xanthine oxidoreductase, deoxyhemoglobin/deoxymyoglobin, and mitochondrial complexes in catalyzing these transformations. Furthermore, we explore how NO generated via this pathway modulates muscle oxygenation through vasodilation and regulation of mitochondrial respiration. The ergogenic potential of dietary nitrate supplementation is discussed in the context of human exercise performance, highlighting the significant controversies, methodological challenges, and sources of inter-individual variability, including genetics and the microbiome. This review aims to provide a comprehensive, chemistry-focused perspective on the nitrate-nitrite-NO pathway, bridging fundamental biochemical mechanisms with their physiological consequences in exercise.