An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate

Abstract

Red blood cells (RBCs) act as O(2)-responsive transducers of vasodilator and vasoconstrictor activity in lungs and tissues by regulating the availability of nitric oxide (NO). Vasodilation by RBCs is impaired in diseases characterized by hypoxemia. We have proposed that the extent to which RBCs constrict vs. dilate vessels is, at least partly, controlled by a partitioning between NO bound to heme iron and to Cysbeta93 thiol of hemoglobin (Hb). Hemes sequester NO, whereas thiols deploy NO bioactivity. In recent work, we have suggested that specific micropopulations of NO-liganded Hb could support the chemistry of S-nitrosohemoglobin (SNO-Hb) formation. Here, by using nitrite as the source of NO, we demonstrate that a (T state) micropopulation of a heme-NO species, with spectral and chemical properties of Fe(III)NO, acts as a precursor to SNO-Hb formation, accompanying the allosteric transition of Hb to the R state. We also show that at physiological concentrations of nitrite and deoxyHb, a S-nitrosothiol precursor is formed within seconds and produces SNO-Hb in high yield upon its prompt exposure to O(2) or CO. Deoxygenation/reoxygenation cycling of oxyHb in the presence of physiological amounts of nitrite also efficiently produces SNO-Hb. In contrast, high amounts of nitrite or delays in reoxygenation inhibit the production of SNO-Hb. Collectively, our data provide evidence for a physiological S-nitrosothiol synthase activity of tetrameric Hb that depends on NO-Hb micropopulations and suggest that dysfunction of this activity may contribute to the pathophysiology of cardiopulmonary and blood disorders.

Fig. 1.

Reaction of deoxyHb with nitrite: Product distribution depends on nitrite concentration. (A) Reaction of deoxyHb (250 μM) with excess (400 μM; Left) or limiting (75 μM; Right) concentrations of nitrite. When excess nitrite is used, the ratio of Hb[Fe(II)NO] to Hb[Fe(III)] approaches 1:1 {and Hb[Fe(III)NO] is detected only at early time points and in trace amounts}. In contrast, with limiting nitrite, Hb[Fe(III)] is in significant excess over Hb[Fe(II)NO], and Hb[Fe(III)NO] accumulates over the time course of the reaction. (B) Hb[Fe(III)]NO production. With high nitrite relative to Hb (400 μM nitrite, 250 μM Hb; ◇), Hb[Fe(III)NO] concentrations rise (over time) until 50% of the hemes in Hb are either ligated by NO or oxidized (sum of all products divided by the heme concentration of 1 mM). Further reaction of nitrite with Hb (to achieve a concentration of products exceeding 50% of hemes) induces the allosteric transition, which is coupled to isomerization of Hb[Fe(III)NO] to SNO-Hb. With limiting nitrite (75 μM nitrite, 1 mM heme; black and white diamonds), Hb[Fe(III)NO] accumulates in T state. Results are presented as mean ± SE of three experiments.

Fig. 2.

Reaction of deoxyHb and nitrite: distribution of products at completion of reaction as a function of initial [NaNO₂]. (A) [NaNO₂] vs. [Fe(III)NO]. Hb[Fe(III)NO] accumulates in reactions with limiting concentrations of nitrite (<250 μM). DeoxyHb, 250 μM. (B) [NaNO₂] vs. [Fe(III)] and [Fe(II)NO]. Limiting (albeit supraphysiological) concentrations of nitrite yield an excess of Fe(III) over Fe(II)NO. DeoxyHb, 250 μM. (C) [NaNO₂] vs. [Fe(III)]/[Fe(II)NO]. The ratio of Fe(III) to Fe(II)NO varies as a function of nitrite concentration. DeoxyHb, 250 μM. Results are presented as mean ± SE of three experiments.

Fig. 3.

Conversion of Hb[Fe(III)NO] to SNO-Hb with transition from T state to R state. The allosteric transition is induced by nitrite (A) or oxygen (B). (A) Fe(III)NO builds up and then isomerizes to SNO-Hb on transition from T state to R state under anaerobic conditions. Product concentrations (at reaction completion) are depicted as a function of percent ligation of all hemes ([DeoxyHb] = 250 μM; [heme] = 4[tetramer]). Nitrite in excess of 250 μM (the concentration producing 50% “ligation,” including both ligated and oxidized hemes) facilitates the allosteric transition from T to R state through an anaerobic buildup of product. (B) Anaerobic values of Hb[Fe(III)NO] concentration are predictive of [SNO-Hb] after oxygenation. Product concentrations are shown as a function of starting nitrite concentration. For [NaNO₂] < 250 μM, Hb[Fe(III)NO] concentration preoxygenation (pre-O₂) correlates directly with [SNO-Hb] after oxygenation (post-O₂). For [NaNO₂] > 250 μM, Hb[Fe(III)NO] concentration decreases to zero and [SNO-Hb] (after oxygenation) plateaus, indicating that an Fe(III)NO intermediate accumulates and then isomerizes to SNO-Hb after the allosteric transition to R state. [DeoxyHb] = 250 μM. Results are presented as mean ± SE of three to four experiments.

Fig. 4.

DeoxyHb and nitrite react immediately to form a SNO-Hb precursor species (A) that produces SNO-Hb upon allosteric transition (B). (A) Samples produced by incubation of 1 μM NaNO₂ with 250 μM deoxyHb for 10 sec yielded a photolysis-chemiluminescence signal representing 565 ± 105 nM NO, which was eliminated in samples desalted with G25. No R state Hb tetramer examined produced a comparable signal after reaction with NaNO₂. Results are presented as mean ± SE; n = 5. (B) Samples produced by mixing 1 μM NaNO₂ with 250 μM deoxyHb as in A were bolused for 10 sec with either CO or O₂, beginning either 10 sec after mixing or after a delay of 1 or 3 min. When samples were bolused with either O₂ or CO beginning 10 sec after mixing, the resultant SNO-Hb concentrations essentially matched SNO precursor concentrations before bolus. However, prolonging anaerobic incubation times before exposure to gas bolus markedly decreased SNO-Hb yields. Results are presented as mean ± SE of five experiments.

Fig. 5.

Cyclic deoxygenation/oxygenation of nitrite and hemoglobin produces SNO-Hb in yields approaching 100%. Rapid deoxygenation of oxyHb (250 μM) in the presence of nitrite (1 μM) followed by reoxygenation produces SNO-Hb effectively as the sole NO product (206 ± 25 nM SNO/224 ± 10 nM total protein-bound NO; open bar). Percent yields of SNO-Hb relative to total proteinbound NO are lower when nitrite is added directly to deoxyHb followed by bolus (as in Fig. 4B) with O₂ (hatched bar) or CO (filled bar). Results are presented as mean ± SE of four to five experiments.