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 beta-93 thiol to NO+ producing center dot SHb(II)NO. 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 center dot SHb(II)NO is formed and that it has the EPR spectrum expected for NO bound to the heme in the beta-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(II)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 degrees 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 beta-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.