INVESTIGATION OF HNO-INDUCED MODIFICATIONS IN VARIOUS SYSTEMS
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Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including small organic molecules, peptides, and a protein. At physiological pH and temperature, relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to investigate sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to sulfinamide detection and used this method to explore sulfinamide hydrolysis. Since HNO-derived modifications may depend on local environment, we have also investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative [RS(O)2NHOH] and a thiosulfonate [RS(O)2SR], presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example with tryptophan resulting in N-nitrosotryptophan formation. We have examined the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. HNO has been shown to enhance cardiac sarcoplasmic reticulum Ca2+ cycling independent of the β-adrenergic pathway. In a collaborative project, the effects of HNO on the cardiac proteins, phospholamban (PLN) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) were investigated.