The case of the missing NO– hemoglobin: Spectral changes suggestive of heme redox reactions reflect changes in NO– heme geometry

Abstract

When low levels of gaseous nitric oxide (NO) are equilibrated with deoxygenated Hb, all NO added can be accounted for in terms of hexacoordinate and pentacoordinate forms of NO-Hb, despite recent reports on NO disappearance from heme groups to form nitroxyl anions or S-nitrosated Hb at low ratios of NO to Hb. We demonstrate that a fraction of the spectral signature of fully nitrosylated (largely hexacoordinate) Hb disappears as the pentacoordinate state forms and reappears when pentacoordinate NO-Hb is reconverted to the hexacoordinate condition. We show that the spectral changes associated with these reversible shifts in NO-heme geometry can be remarkably well approximated as variations in the contributions from fully nitrosylated Hb and oxidized Hb (MetHb). As a result, increases in the level of pentacoordinate NO-Hb that occur at low NO to Hb ratios can be misinterpreted as increases in MetHb levels associated with NOdependent heme oxidation. Conversely, any decrease in levels of pentacoordinate NO-Hb can be misinterpreted as a disappearance of MetHb associated with NO-dependent heme reduction. Transitions between pentacoordinate and hexacoordinate forms of NO-Hb with spectral changes suggestive of changes in levels of heme-bound NO are sensitive to the protein's quaternary conformation and can be brought about by alterations in anion levels or the degree of heme saturation with either O 2 or NO. O rganic phosphates and low pH, which stabilize the lowaffinity T-state of Hb, bring about differences in the interactions of Hb and NO that differ from those observed at high pH, where Hb's R-state conformation is favored (1-6). The literature is less clear about differences resulting from variations in levels of heme ligation with NO, a ligand with extremely high affinity for ferrous heme (7-9).A direct influence of the NO to Hb ratio on NO interactions with Hb was suggested by Gow and Stamler (10), who hypothesized that the NO-Hb level is significantly depleted at low NO to Hb ratios by redox-driven formation of MetHb and the nitroxyl ion (NO Ϫ ) in the absence of oxygen and by formation of S-nitrosated Hb (SNO-Hb) at the expense of NO-Hb in the presence of oxygen. In support of these concepts, these authors reported changes in the NO-Hb spectrum seemingly coupled with changes in MetHb and SNO-Hb levels.To explore the interactions of deoxy Hb with low to saturating levels of NO, we sought to quantitatively account for all NO introduced into large-volume tonometers containing deoxy Hb. The data obtained argue against the hypothesis of redoxmediated depletion of NO-Hb at low ratios of NO to Hb (10) and show instead that NO is bound stoichiometrically to deoxy Hb from low to high degrees of heme nitrosylation. Here we present an alternative view of the reactions of NO and deoxy Hb at varied NO to Hb ratios (supported by electronic absorption and EPR spectroscopies) that is based on iron coordination shifts between hexacoordinate and pentacoordinate forms of NO-Hb rather than on redox-or oxygen-linked alterations of N...

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

“…In previous studies, we included in the basis of spectral components all Hb spectra that could be reasonably expected under the given experimental conditions. However, the calculated best-fit spectra still show small systematic deviations from experimental ones, both in our studies and related studies by others (28,29). In estimating the precision of the fitted parameter values, we included effects of parameter correlation and systematic error of the model and, thus, reported conservative error estimates of Ϸ10 M (a concentration exceeding the NO-Hb species of physiological relevance; refs.…”

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions

“…(2)) [20]. The production of SNOHb accompanying exposure of Hb to nitrite has since been observed by two other research groups [25]; its formation in the aftermath of the oxygenation of Hb Fe(II)NO species has also been observed in several laboratories [26,27].…”

“…Luchsinger, D.J. Singel, unpublished results) [30] to determine the amounts of six-coordinate, and five-coordinate hemeFe(II)NO complexes, respectively [27]. The particular methods that we use to improve the performance of the spectral decomposition have been noted previously.…”

Molecular Controls of the Oxygenation and Redox Reactions of Hemoglobin

“…Exposure of Hb to NO and formation of HbNO in the absence of O 2 does not trigger SNO-Hb formation in either T-or R-states (39), but transfer of NO + from intraerythrocytic S-nitrosoglutathione to the reactive thiol group of b93 Cys in the R-state of Hb could occur in the lungs, forming a limited amount of SNO-Hb. SNO-Hb could also result from formation of nitrosating agents such as NO x when NO is generated from nitrite in the presence of O 2 (7,21,39). Deoxygenation of SNO-Hb in the microvasculature, accompanied by the reverse R-to-T process, was hypothesized to cause the release of NO equivalents.…”