Nitrite and nitrate chemical biology and signalling

Abstract

Inorganic nitrate (NO3 - ), nitrite (NO2 - ) and NO are nitrogenous species with a diverse and interconnected chemical biology. The formation of NO from nitrate and nitrite via a reductive 'nitrate-nitrite-NO' pathway and resulting in vasodilation is now an established complementary route to traditional NOS-derived vasodilation. Nitrate, found in our diet and abundant in mammalian tissues and circulation, is activated via reduction to nitrite predominantly by our commensal oral microbiome. The subsequent in vivo reduction of nitrite, a stable vascular reserve of NO, is facilitated by a number of haem-containing and molybdenum-cofactor proteins. NO generation from nitrite is enhanced during physiological and pathological hypoxia and in disease states involving ischaemia-reperfusion injury. As such, modulation of these NO vascular repositories via exogenously supplied nitrite and nitrate has been evaluated as a therapeutic approach in a number of diseases. Ultimately, the chemical biology of nitrate and nitrite is governed by local concentrations, reaction equilibrium constants, and the generation of transient intermediates, with kinetic rate constants modulated at differing physiological pH values and oxygen tensions. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc.

© 2018 The British Pharmacological Society.

Figures

Figure 1

Myriad chemistries of nitrite (NO2 −) leading to protective and signalling effects. Nitrite can be reduced to NO (mechanisms discussed below), which nitrosylates a ferrous haem in sGC, triggering the production of cGMP from GTP. cGMP triggers a signal cascade resulting in vasodilation. Alternatively, nitrate can be oxidized (conditions described below) to yield the oxidant NO2 •. NO2 • reacts with a number of targets, but under conditions of excess NO2 •, can participate in nitration chemistry of fatty acids (FAs). The generated nitro‐fatty acids are electrophilic and modify critical redox active thiols to mediate signalling, for example, by activating transcription factors like Nrf‐Keap1. Nitrite can also be dehydrated catalytically or in the presence of acid (vide infra) to generate the strong nitrosating agent N2O3. N2O3 can react with nucleophiles such as thiols resulting in S‐nitrosothiol (RSNO) formation. Here, this activity is depicted on a critical thiol of complex I of the mitochondrial electron transport chain, preventing formation of damaging superoxide (especially under I/R injury). N2O3 can also disproportionate into NO2 • and NO. Dotted arrows represent reactions that are also catalysed.

Figure 2

NiR activity of haemoproteins in O‐nitrito nitrite coordination (i.e. Mb and Hb). Addition of a proton either directly from the histidine (shown) or water yields the nitrous acid bound species. After protonation and subsequent inner‐sphere electron transfer from the ferrohaem to the bound nitrous acid, NO dissociates leaving the hydroxo‐ferrihaem complex which can then be protonated to the aquomet form.

Figure 3

Simulated data representing the NiR activity of deoxymyoglobin and deoxyhaemoglobin under anoxic conditions, specifically tracing the formation of nitrosylated ferrohaem (Equation (6)) over time. Mb, which has a constant kNiR, behaves in an exponential manner. Hb exhibits autocatalysis and a sigmoidal shape: tetrameric T‐state (blue zone) deoxyHbII reduces nitrite, generating a methaem (3+) and an equivalent of NO. The NO binds a vacant ferrohaem on the same or different tetramer to form a nitrosylated ferrohaem species. Each of these new species stabilizes the R‐state (red zone), which has a higher bimolecular rate constant than the T‐state, resulting in an increase in apparent rate and propagating nitrite reduction. As the vacant reactive ferrohaem sites are filled, the rate of nitrosylated‐ferrohaem formation drops off.

Figure 4

Various mechanisms of non‐enzymically induced redox cycling ferrihaem (blue‐centres) to ferrohaem (red‐centres). The top path represents reductive nitrosylation, where NO displaces water from the aquomet‐globin generating a nitrosyl‐ferrihaem/nitrosonium‐ferrohaem (shown). A nucleophile such as hydroxide/water (path c) generates nitrite, whereas when nitrite is the nucleophile (path b), N2O3 is generated. Importantly, other nucleophiles such as thiolates can participate in this chemistry. Similarly, the nitrite anhydrase mechanism (path a) represents a situation where nitrite binds the methaem first. The subsequent nitrito‐ferrihaem complex (here depicted as O‐nitrito, but the N‐nitro is possible) exhibits some radical NO2 •‐ferrohaem character, which can readily react with NO to generate diffusible N2O3.

Figure 5

The nitrate–nitrite–NO pathway and simplified nitrite chemical biology in the vasculature. The nitrate–nitrite–NO path is a complementary route for NO generation in the vasculature that occurs under hypoxic conditions, when canonical NO generation at normoxia from eNOS (upper left) is less effective. Nitrate is ingested (upper right) from various foods including beetroot and leafy greens. Oral bacteria in the salivary glands reduce nitrate to nitrite, which is subsequently swallowed, traversing the oesophagus and into the acidic gastric fluids and into GI tract (hollow arrowheads). Some of the nitrite (as nitrous acid) may dehydrate and disproportionate (Equations (3) and (4) in text) to generate NO in the stomach. Orally swallowed nitrite has been suggested to be bioactivated in the stomach under non‐enzymic acidic conditions. Nitrite may also escape the GI tract and enter the circulation (bottom right). Numerous reactions occur in RBCs (centre left), especially at the point of artery‐to‐vein transport (Hb is ~50% O2‐saturated or P50) where the nitrite reduction rate by deoxyHb is maximized. NiR activity yields metHb and an equivalent of NO (Equation (6)). metHb reacts with nitrite to form a radical NO2 •‐bound ferrohaem, which reacts rapidly with NO to generate N2O3 (Figure 4, path a), responsible for RSNO formation (Equation (9)). N2O3 is one mechanism by which NO is proposed to escape from the RBC and generate NO (Equation (4)) in the smooth muscle. NO may also be autocaptured by deoxyHb generating ferrous nitrosyl‐Hb (Equation (7), not shown) and subsequently released via oxidative nitrosylation (Equations (11) and (12), not shown), or NO may react with oxyHb to liberate nitrate (Equation (2)). The nitrate is secreted from the plasma into the salivary glands (upper right), starting the process anew. XO and eNOS found in both endothelial cells and the surface of RBCs have been suspected to generate NO from nitrite, but only under hypoxic and more acidic conditions (bottom left). Finally, nitrite is generated by the copper plasma protein ceruloplasmin from NO, preventing dioxygenation to nitrate or reductive nitrosylation by ferrihaems (not shown).