A New Paramagnetic Intermediate Formed during the Reaction of Nitrite with Deoxyhemoglobin

Abstract

The reduction of nitrite by deoxygenated hemoglobin chains has been implicated in red cell induced vasodilation, although the mechanism for this process has not been established. We have previously demonstrated that the reaction of nitrite with deoxyhemoglobin produces a hybrid intermediate with properties of Hb(II)NO+ and Hb(III)NO that builds up during the reaction retaining potential NO bioactivity. To explain the unexpected stability of this intermediate, which prevents NO release from the Hb(III)NO component, we had implicated the transfer of an electron from the β-93 thiol to NO+ producing •SHb(II)NO. In order to determine if this species is formed and to characterize its properties, we have investigated the electron paramagnetic resonance (EPR) changes taking place during the nitrite reaction. The EPR effects of blocking the thiol group with N-ethyl-maleimide and using carboxypeptidase-A to stabilize the R-quaternary conformation have demonstrated that •SHb(II)NO is formed and that it has the EPR spectrum expected for NO bound to the heme in the β-chain plus that of a thiyl radical. This new NO-related paramagnetic species is in equilibrium with the hybrid intermediate “Hb(II)NO+ ↔ Hb(III)NO” thereby further inhibiting the release of NO from Hb(III)NO. The formation of an NO-related paramagnetic species other than the tightly bound NO in Hb(II)NO was also confirmed by a decrease in the EPR signal by −20°C incubation, which shifts the equilibrium back to the “Hb(II)NO+ ↔ Hb(III)NO” intermediate. This previously unrecognized NO hemoglobin species explains the stability of the intermediates and the buildup of a pool of potentially bioactive NO during nitrite reduction. It also provides a pathway for the formation of β-93 cysteine S-nitrosylated hemoglobin [SNOHb:S-nitrosohemoglobin], which has been shown to induce vasodilation, by a rapid radical-radical reaction of any free NO with the thiyl radical of this new paramagnetic intermediate.

Evidence for a cysteine-mediated mechanism of excitation energy regulation in a photosynthetic antenna complex

“…6) indicate that the organic radical seen in the WT protein effectively disappears upon mutation of either or both cysteine residue(s). This EPR signature is consistent with previously published spectra of thiyl radicals (30)(31)(32)(33).…”

S-nitrosylation therapy to improve oxygen delivery of banked blood

“…A role for nitrite in relaxation by RBCs has also gained credence. However, nitrite is a nuance of the SNO-based mechanism (16,24) [not a rejection of it as originally put forth by Cosby et al (50)] because nitrite acts as a precursor to SNO-Hb formation (16,17,27,28,51). Indeed, initial claims that free NO derived from nitrite could escape the hemes in Hb to elicit vasodilation independently of SNOs (50) have not been reproduced (even by the authors themselves) (52)(53)(54), and were likely an in vitro artifact of reagents added to the bioassay system (16,24,52,53).…”

“…Specifically, it is proposed that the conformational changes incurred upon binding and release of O 2 from the hemes are intimately linked to binding and release of NO from cysteine residues in Hb, and that the released NO is liberated from RBCs in the form of bioactive S-nitrosothiols (SNOs). There is further appreciation that NO derived from nitrite may participate in RBC vasodilation through its conversion into SNOs (16,17,27,28). [Note, however, that NO itself cannot escape sequestration by excess hemes of Hb (24).]…”

Hemoglobin Redox Reactions and Red Blood Cell Aging

“…These metastable intermediates retain NO bioactivity, but are not quenched by reacting with Hb or superoxide. These intermediates, thus, provide a pool of potentially bioactive NO, which can be released to the vasculature when needed (84,91,92).…”