Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO-adducts of ferrous globins

Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O2, and we have previously reported that deoxymyoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy, and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yields by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O2−FeII adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in 1H NMR at ca. 15 ppm that splits into doublets for H15NO adducts. The 1H and 15N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in the reduction rates of the NO adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UV−vis, NMR, and...