In site directed spin labeling, a nitroxide side chain is introduced at a selected site in a protein; the most commonly used is a disulfide-linked side chain designated R1. The EPR spectra of R1, and the interspin distance between pairs of R1 residues as determined by dipolar EPR spectroscopy, encode a wealth of information on the protein structure and dynamics. However, to extract this information requires structural and dynamical models of the R1 side chain, i.e. the favored rotamers, the intraresidue interactions that stabilize them, and the internal modes of motion. X-ray crystal structures of R1 in proteins have revealed a set of preferred rotamers in the crystal lattice. To identify the intraresidue interactions that stabilize particular rotamers of R1 in the absence of interactions with nearby sidechains in a helix, and to evaluate models for the internal motion of the side chain, quantum mechanical calculations were performed on a relevant fragment of R1 in a ten-residue α-helix. Relative rotamer energies were determined in the gas phase, and solvation energies were estimated from a continuum solvent model that includes both electrostatic and hydrophobic contributions. The results identified preferred rotamers that are in agreement with the X-ray crystallographic studies. The rotamers are apparently stabilized by intra-residue sulfur-backbone interactions, suggesting that the preferred rotamers may be the same at all solvent-exposed helix sites.

Nitroxyl (HNO), the one-electron reduced and protonated congener of nitric oxide (NO), is a chemically unique species with potentially important biological activity. Although HNO-based pharmaceuticals are currently being considered for the treatment of chronic heart failure or stroke/transplant-derived ischemia, the chemical events leading to therapeutic responses are not established. The interaction of HNO with oxidants results in the well-documented conversion to NO, but HNO is expected to be readily reduced as well. Recent thermodynamic calculations predict that reduction of HNO is biologically accessible. Herein, kinetic analysis suggests that the reactions of HNO with several mechanistically distinct reductants are also biologically feasible. Product analysis verified that the reductants had in fact been oxidized and that in several instances HNO had been converted to hydroxylamine. Moreover, a theoretical analysis suggests that in the reaction of HNO with thiol reductants, the pathway producing sulfinamide is significantly more favorable than that leading to disulfide. Additionally, simultaneous production of HNO and NO yielded a biphasic oxidative capacity.