Nitrosyl Hydride (HNO) as an O₂ Analogue: Long-Lived HNO Adducts of Ferrous Globins

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 deoxy myoglobin 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 yield 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 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 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 UVvis, NMR and EPR analysis. The two peaks of HNO-hHb 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. KeywordsHNO; nitroxyl; nitrosyl hydride; dioxygen; globins; heme oxygenase Nitrosyl hydride (HNO), the protonated form of nitroxyl anion (NO − ), has distinct physicochemical properties from its congener nitric oxide (NO), much of which has been defined only recently. 1,2 The anionic form is isoelectronic with dioxygen and exists as a triplet, 3 NO − above pH 12; at lower pH the singlet 1 HNO dominates, but is susceptible to rapid † This research was supported by the National Science Foundation (PJF CHE-0100774) and the National Institutes of Health (PJF 1R21ES016441-01).*To whom correspondence should be addressed. E-mail: pfarmer@uci.edu. . SUPPORTING INFORMATION AVAILABLE. Experimental details available include descriptions of peak fitting for isoforms of native legHb mixtures, timecourse UVvis spectra during the formation of HNO-hHb, EPR characterization of ferrous NO adduct impurities in HNO adduct samples and data for initial rate analysis of HNO trapping by deoxy Mb and hHb. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 9. The rate of this dimerization has been reported as 8 × 10 6 M −1 s −1,5 and thus severely limits the lifetime and concentration of HNO generated in solution.(1)HNO is the simplest analogue of alkylnitroso compounds, RNO, long known to bind to ferrous heme proteins. 6 Mansuy and coworkers were the first to describe the binding of RNO compounds to ferrous globins myoglobin (Mb) and human hemoglobin (hHb), 7 as well as to make the analogy of RNO bindi...

A comparison of the chemistry associated with the biological signaling and actions of nitroxyl (HNO) and nitric oxide (NO)

“…Probably the most distinguishing and diagnostic characteristic of the ferrous myoglobin HNO complexes is the 1 H NMR resonance for the bound HNO at about 15 ppm. The HNO adducts with ferrous myoglobin are inherently stable under strict anaerobic conditions and can be stored for extended periods of time without degradation [29]. However, exposure of the ferrous HNO myoglobin complex to O 2 results in rapid ferric myoglobin formation and exposure to NO or NO 2 − gives a ferrous-NO complex [28].…”

“…However, exposure of the ferrous HNO myoglobin complex to O 2 results in rapid ferric myoglobin formation and exposure to NO or NO 2 − gives a ferrous-NO complex [28]. Interestingly, it appears that the ferrous HNO myoglobin adduct can react with excess HNO to also give the NO adduct (Reaction 8) [29]. Of particular note is the possibility that a ferrous-HNO complex in sGC could be converted to the ferrous-NO complex by a variety of species (i.e.…”

A singular value decomposition approach for kinetic analysis of reactions of HNO with myoglobin

“…Free HNO is quite reactive and short-lived in aqueous solution [12], but we have shown that an HNO adduct of ferrous myoglobin, HNO-Mb can be formed by reduction of NO-Mb [13,14], by trapping of free HNO by deoxymyoglobin (deoxyMb or Mb-Fe II ) [15], or by treatment of metmyoglobin (metMb or Mb-Fe III ) with salts of nitrite and borohydride [16]. This stable ferrous heme adduct has been characterized by 1 H NMR [17], 1 H-15 N heteronuclear single quantum coherence (HSQC) [18], as well as resonance Raman and X-ray absorption spectroscopies [19]. As HNO is isoelectronic with O 2 and forms stable diamagnetic adducts with ferrous hemoglobins from various species, we have suggested its use as an O 2 analog [18].…”

“…This stable ferrous heme adduct has been characterized by 1 H NMR [17], 1 H-15 N heteronuclear single quantum coherence (HSQC) [18], as well as resonance Raman and X-ray absorption spectroscopies [19]. As HNO is isoelectronic with O 2 and forms stable diamagnetic adducts with ferrous hemoglobins from various species, we have suggested its use as an O 2 analog [18]. Most recently, we have shown that HNO may replace O 2 during turnover of a non-heme Mn dioxygenase enzyme, quercetin dioxygenase; this nitroxygenase activity results in the regioselective incorporation of the N atom derived from HNO within the enzymatic product [20].…”

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

“…15 ppm, well away from other protein resonances, which allowed structural characterization of the heme pocket by 2D NOE and COSY experiments (10). Other ferrous globins also rapidly trap free HNO to directly yield stable HNO adducts, identifiable by changes in electronic absorbances as well as unique 1 H and 15 N NMR resonances due to the nitrosyl hydride (11).…”