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...

The formation and interconversion of nitrogen oxides has been of interest in numerous contexts for decades. Early studies focused on gas-phase reactions, particularly with regard to industrial and atmospheric environments, and on nitrogen fixation. Additionally, investigation of the coordination chemistry of nitric oxide (NO) with hemoglobin dates back nearly a century. With the discovery in the early 1980s that NO is biosynthesized as a molecular signaling agent, the literature has been focused on the biological effects of nitrogen oxides, but the original concerns remain relevant. For instance, hemoglobin has long been known to react with nitrite, but this reductase activity has recently been considered to be important to produce NO under hypoxic conditions. The association of nitrosyl hydride (HNO; also commonly referred to as nitroxyl) with heme proteins can also produce NO by reductive nitrosylation. Furthermore, HNO is considered to be an intermediate in bacterial denitrification, but conclusive identification has been elusive. The authors of this article have approached the bioinorganic chemistry of HNO from different perspectives, which have converged because heme proteins are important biological targets of HNO.

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The synthesis, characterization and spectral properties of six new meso-aryl core modified sapphyrins are described. An efficient approach involving an acid catalyzed condensation of bithiophene diol 1 and modified tripyrranes 2a-2e allows preparation of the desired meso-aryl sapphyrins in 16-36% yield. The product distribution and the isolated yield were found to be dependent on the nature of the acid catalyst (Lewis acid or protic acid) and its concentration. Protic acid catalyst exclusively gave the expected sapphyrins while two additional products, an 18π tetraphenylporphyrin and a 26π modified rubyrin, were isolated under Lewis acid catalysis. An analysis of proton NMR and absorption spectral data suggests that in free base sapphyrins, the heterocyclic ring opposite to the bithiophene unit is inverted as in N-5 meso-aryl sapphyrin and the degree of inversion is dependent on the nature of the heterocyclic ring. The energy optimized structure calculated from the semi-empirical method substantiates such a conclusion. Protonation of sapphyrins generates respective mono-and dications and the heterocyclic ring retains an inverted structure in contrast to normal N-5 sapphyrins. The triplet excited lifetimes for free base and protonated derivatives are similar both under argon saturated and air equilibrated conditions, indicating that the triplet state quenching by oxygen is minimal. Cyclic voltammetric studies reveal easier reductions and harder oxidations relative to meso-aryl porphyrins and the ∆ redox observed for 3d suggests significant reduction of the HOMO-LUMO energy gap consistent with the large red shift observed for the Soret band.

Lipophilic hexaporphyrin free-base and copper-metalated assemblies supported on a Sn6O6 core have been synthesized and characterized. The nuclease activity of the copper derivative has been studied.