A New Paramagnetic Intermediate Formed During The Reduction Of Nitrite By Deoxyhemoglobin

Nitric oxide (NO) plays a crucial role regulating vascular tone and blood flow. Recent studies have shown that RBCs can take up nitrite at reduced oxygen pressures to be reutilized by deoxyhemoglobin for the production of nitric oxide. However, it is still necessary to explain how NO bypasses hemoglobin scavenging. Our previous studies have suggested the reduction of nitrite back to NO by deoxyhemoglobin via intermediates that retain NO bioactivity. One of the intermediates characterized was a hybridized species with properties of Hb(II)NO+ and Hb(III)NO. The unexpected stability of this intermediate has been attributed to the transfer of an electron from the β-93 thiol of hemoglobin to NO+ producing •SHb(II)NO. The formation of this paramagnetic intermediate (•SHb(II)NO) was confirmed by studying the electron paramagnetic resonance (EPR) changes that take place during nitrite reduction by deoxyHb in comparison with deoxyHb treated with N-ethyl-maleimide (blocks the β-93 thiol group of hemoglobin) and carboxypeptidase A (removes C-terminus of hemoglobin), both of which stabilize the R-quaternary conformation of hemoglobin. The spectral changes observed were attributed to the equilibrium of the hybridized diamagnetic species with a paramagnetic species with an EPR spectrum that reflects that of NO bound to the heme in the β-chain plus that of a thiyl radical, •SHb(II)NO. Incubation studies at −20°C demonstrate that the intensity of this paramagnetic species decreases at this low temperature indicating a shift back to the hybrid intermediate, “Hb(II)NO+ ↔ Hb(III)NO”. This new paramagnetic intermediate species (•SHb(II)NO) explains the stability of the hybridized intermediate and the slow release of NO during the nitrite reduction. The involvement of a thiyl radical, at the same time provides a pathway for the formation of SNOHb that can also be a source for NO bioactivity.