Oxidation of human nitrosylhemoglobin monitored by UV-Vis and EPR spectroscopies : detection of products and intermediates

Abstract

Once viewed only as a toxic free radical, nitric oxide (NO) has been established as an essential and ubiquitous signaling and regulatory molecule in biological systems. Notably, NO was identified as the endothelium-derived relaxing factor (EDRF) in the blood. NO is capable of complex redox chemistry and interaction with a host of protein families. Among these proteins is hemoglobin (Hb) which can interact with NO at the level of the heme and can bind NO at Cys93 on its â subunit to form S-nitrosylated Hb (SNO-Hb). NO bound as SNO-Hb is chemically labile and thus preserves bioavailability of NO. However, when NO reacts with oxyHb or deoxyHb NO bioavailability is quenched by conversion to nitrate or by tightly binding the heme, respectively. Therefore, the question is raised as to how NO can be EDRF in the presence of such high Hb concentrations in the blood. One way NO availability can be preserved is by exploiting the redox chemistries of both Hb and NO. Human Hb(NO)4 oxidation by K3Fe(CN)6 was studied and products and intermediates were identified by UV-Vis and EPR spectroscopies. Periodically, samples were withdrawn from the reaction mixture for nitrosylation product and/or EPR analysis. Reaction spectra converted to heme species concentration vs. time plots through leastsquares fitting of five basis spectra. These data were then utilized to generate a de minimis model of the oxidation reaction. We demonstrate that the oxidation of Hb(NO)4 by K3Fe(CN)6 1) leads to the production of SNO-Hb, 2) occurs preferentially at the â heme, and 3) proceeds through an HbFeIIINO intermediate.