The functional nitrite reductase activity of the heme-globins

Abstract

Hemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N(2)O(3)), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart.

Figure 1

The sigmoidal “apparent zero order” behavior of the nitrite reaction with deoxyhemoglobin. Deoxyhemoglobin (50 μM) reaction with nitrite (10 mM) at pH 7.4 and 37°C. (A) Time-resolved absorption spectra were deconvoluted to determine the percentage of each species as a function of time. Deoxyhemoglobin is observed to form equal amounts of methemoglobin and iron-nitrosyl-hemoglobin. Deviation from first-order behavior is evident in the curve for decay of deoxyhemoglobin, having a sigmoidal shape. In experiments with excess nitrite, the spectrum of nitrite bound to methemoglobin (nitrite-methemoglobin) should be included. (B) The instantaneous rate of the reaction shown in panel A where the negative of the slope of the decay curve for deoxyhemoglobin is plotted as a function of time. The figure is reproduced from Grubina et al with permission.

Figure 2

(A) Allosteric autocatalysis. Reaction of nitrite with deoxyhemoglobin exhibits R-state catalysis. Tetrameric T-state deoxyHb reduces nitrite to NO, generating a met-heme (3⁺) and an iron-nitrosyl-heme (Fe⁺²-NO), on the same or different Hb tetramers, which stabilize the tetramer(s) in the R state. Increasing R-state character is associated with a higher bimolecular rate constant for nitrite reduction. As a result, ferrous deoxyhemes on these R-state stabilized tetramers react with nitrite faster than those on T-state stabilized tetramers, thereby exponentially propagating nitrite reduction and R-state stabilization. This process therefore represents a unique allosteric autocatalytic reaction mechanism. Please note that we are showing the bimolecular rate constant, not the overall reaction rate, and that in the case of hemoglobin, the overall hemoglobin rate constant is not constant, but changes with the T-to-R allosteric transition. The overall rate constant is dependent on the intrinsic reactivity of nitrite with heme and is highest in R state. The actual rate of a second-order reaction is determined by the concentration of deoxyheme multiplied by the concentration of nitrite and multiplied by the bimolecular rate constant. Panel A is reproduced from Grubina et al with permission. (B) Distribution of R and T ligand populations modulates nitrite reduction. The fractions of R and T oxygen-liganded species are plotted as a function of oxygen saturation. The quaternary state (R or T) is indicated and the number of ligands bound to each tetramer is indicated by the subscript, so that R₃ indicates an R-state conformation with 3 oxygen ligands bound. The fraction of each species was calculated using the MWC Perutz 2 state model with the value of c set at 0.015, where c is the ratio of equilibrium-binding constants for T (taken as 1/77 mm Hg) and R states. The ratio L = T₀/R₀ was taken as 10⁵. The fractions of some intermediate species are so small that they do not appear on the graph. (C) Contribution of quaternary states to nitrite reaction rate. The reaction rates of nitrite with deoxygenated Hb are plotted as a function of oxygen saturation for cases where the product of the nitrite and Hb concentrations are 10⁻⁶ M². At each oxygen saturation the rate of the reaction was calculated as [nitrite] × {k_t(4[T₀] + 3[T₁] + 2[T₂] + [T₃]) + k_R(4[R₀] + 3[R₁] + 2[R₂] + [R₃]), where the square brackets refer to Hb concentrations. Here, k_R/k_T was rounded to 100 and k_T was set to 0.2 M⁻¹s⁻¹. The contribution by R-state and T-state molecules was obtained by calculating the products of k_R and k_T separately (so, for example, the R-state contribution is [nitrite] × k_R(4[R₀] + 3[R₁] + 2[R₂] + [R₃])).

Figure 3

The N₂O₃-forming reaction of nitrite and hemoglobin may regulate export of NO from the erythrocyte. Hemoglobin deoxygenation (purple) occurs preferentially at the submembrane of the red blood cell as it traverses the arteriole. Nitrite reacts with deoxygenated hemoglobin to make methemoglobin and NO. Methemoglobin binds nitrite to form an adduct with some Fe⁺²-NO₂ character (Hb-NO₂^•). This species reacts quickly with NO, forming N₂O₃, which can diffuse out of the red cell, later forming NO and effecting vasodilation and/or forming nitrosothiols (SNO). Low-molecular-weight nitrosothiols may contribute to exportable vasodilatory activity. The Hb abbreviation indicates ferrous deoxyhemoglobin (Fe⁺²). Figure is reproduced from Basu et al with permission.

Figure 4

Biochemistry of nitrite-hemoglobin hypoxic vasodilation along the A1 to A5 arterioles. There exists a steady state anatomic location within the circulation from artery to vein that has the greatest concentration of R₃ tetramers (orange line bottom panel), which possess the maximal nitrite reductase activity. At this location, there would always exist an equilibrium rate constant for nitrite reduction (red line top panel and green line bottom panel) and an equilibrium concentration of nitrite and deoxyhemes (maximized in R₃ tetramer). As soon as one red cell moves downstream a new one would replace it, thus preserving the concentration of nitrite and R₃ hemoglobin at that anatomic position. Thus, there will be an increased nitrite reductase rate and increased NO concentration surrounding the blood vessel. The NO concentration should increase in a bell curve distribution from artery to vein according to the predicted rate for nitrite reduction. The anatomic position of this equilibrium NO concentration will be responsive to tissue metabolism and oxygen consumption by moving the R-to-T transition upstream or downstream. Note that the rate of a second-order reaction is determined by the product of the concentration of 2 reactants and the bimolecular rate constant. In this case, the nitrite concentration changes only a little as hemoglobin deoxygenates, the deoxyhemoglobin concentration increases dramatically (blue line top panel, brown line bottom panel), whereas the bimolecular rate constant decreases dramatically (red line top panel, green line bottom panel) as hemoglobin goes from the R-to-T conformation. So the product of bimolecular rate constant and deoxyheme concentration peaks from 60% to 40% hemoglobin oxygen saturation when the most R₃ tetramers are present. Figure was modified and reproduced from Gladwin et al^, with permission.