Stable nitroxyl radicals as pH, thiol and electron transfer probes

Abstract

In this review article original applications of stable nitroxyl radicals (SNRs) developed by the author with his students and colleagues are presented. ESR (electron spin resonance) spectra of SNRs of the imidazoline and imidazolidine types display high pH sensitivity. Mechanisms of proton exchange in such SNRs were studied in detail and made possible their wide application as pH probes (range of pH 0-13.5) in a variety of chemical and biological systems, including in vivo assays. A stable biradical, bis (2,2,5,5-tetramethyl-3-imidazoline-1-oxyl-4-il)-disulfide, was synthesized and used to monitor thiol-disulfide exchange with free thiols in solution. This approach permitted us to develop an extremely sensitive, quantitative and noninvasive method for determining the concentrations of glutathione and cysteine in living cells. The biradical was used to clarify the topography of cysteines in enzymes and to study their involvement in folding and stability of some proteins. We proposed and implemented a novel ESR approach to study electron transfer: the kinetics of photooxidation of an SNR (with concomitant production of an ESR-silent oxoammonium ion) by a Ru(III)-bipyridyl complex. Recovery of the ESR signal was observed when illumination was terminated. The rate of recovery depends strongly on the presence of organic electron donors, including some amino acids, and on pH. We used site-directed spin-labeled mutants of bacteriorhodopsin, to which a Ru(III) complex was also bound at a well-defined locus, to show the applicability of the proposed method to study the electron transfer and effects of local neighboring and water penetration in proteins. *** DIRECT SUPPORT *** A04RK041 00002