The Reaction between Nitrite and Oxyhemoglobin

Abstract

The nitrite anion (NO 2 ؊ ) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO 2 ), and hydrogen peroxide (H 2 O 2 ) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H 2 O 2 and NO 2 were thought to be integral to both the initiation and propagation steps, H 2 O 2 now only plays a role as an initiator species, and NO 2 only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H 2 O 2 and NO 2 in the reaction mechanism for the in vivo reactivity of nitrite are discussed.Elevated levels of nitrite in blood can trigger the oxidation of hemoglobin, leading to methemoglobinemia (1, 2). The mechanism of nitrite-dependent oxidation of deoxyhemoglobin (deoxyHb) 2 under anaerobic conditions, and its physiological potential in hypoxic vasodilation has recently been established (3-5). However, the mechanism of reaction between oxyhemoglobin (oxyHb) and nitrite has not yet been fully elucidated. Existing mechanistic approaches, starting with the earliest observations of Gamgee in 1868 (6), were recently reviewed (7). The end products of the reaction are methemoglobin (metHb) and nitrate. The kinetics of this reaction, when nitrite is in excess, are complex and exhibit an initial slow phase (often referred to as the lag phase) that accelerates into a rapid phase (referred to as the propagation phase) of oxidation. This reaction profile has been modeled as an autocatalytic, free radical chain reaction. The chain carrier radical and the autocatalytic steps have been variously hypothesized. Doyle et al. (8) found that hydrogen peroxide (H 2 O 2 ) accelerated oxyHb decay, whereas both catalase and superoxide dismutase elongated the half-time of the reaction. Based on these observations they suggested a mechanism according to which H 2 O 2 and superoxide played crucial roles. At the same time, Kosaka et al. (9) observed that catalase could extend the slow phase, but observed no effect of superoxide dismutase. In addition, they detected a transient protein free radical by EPR spectroscopy, and proposed a model with the intermediacy of H 2...

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