Abstract
Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O(2), 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 O(2)-Fe(II) adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in (1)H NMR at ca. 15 ppm that splits into doublets for H(15)NO adducts. The (1)H and (15)N 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 EPR analysis. The two peaks of the HNO-hHb adduct have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes.