Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological fu nctions. Some applications of electron paramagnetic resonance (EPR) spectro scopy are reviewed, for observing nitrosyl species in biological systems. N itrite has long been used as a food preservative owing to its bacteriostati c effect on spoilage bacteria. Nitrosyl complexes such as sodium nitropruss ide, which are added experimentally as NO-generators, themselves produce pa ramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl comple xes such as (RS)(2)Fe(NO)(2). Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cel ls by the immune system. The targets include iron-sulfur proteins. It appea rs that species derived from nitric oxide such as peroxynitrite may be resp onsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in v ivo from hemoglobin, in conditions such as experimental endotoxic shock. Th is has been used to follow the course of production of NO by macrophages. W e have examined the effects of suppression of NO synthase using biopterin a ntagonists. Another method is to use an injected NO-trapping agent, Fe-diet hyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way w e have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an i ndication for the involvement of nitric oxide or a derived species in the d amage to this organ. (C) 1998 Elsevier Science B.V. All rights reserved.