Electron paramagnetic resonance studies of nitrosyl ferrous heme complexes. Determination of an equilibrium between two conformations.

Ferrous hemeproteins and inorganic heme complexes react with nitric oxide to form six-coordinate paramagnetic complexes. These complexes exhibit strong EPR signals centered near g = 2.0 which are easily observed in frozen solution. We have obtained EPR spectra of the nitrosyl adducts of Fe(II) protoporphyrin IX-imidazole in dimethylsulfoxide and in dimethylformamide, and of myoglobin, cytochrome c, and cytochrome c oxidase in buffered solution over a range of temperatures from 30-180 K. All but the last of the complexes exhibit temperature-dependent EPR spectra. This temperature dependence could be due to motional/relaxation effects or to a chemical equilibrium. To resolve this matter, we have used the technique of factor analysis to deconvolute the temperature-dependent EPR spectra. By this method we have found that all of the spectra for any given complex can be reproduced by adding together varied amounts of two signals, demonstrating that the variation of the EPR spectra with temperature is due to an equilibrium between two species. The two species differ in enthalpy by no more than about 2 kcal/ mol. We interpret the observed signals as arising from two six-coordinate conformers of the nitrosylheme e n i trogen base complexes, differing primarily in the position of the iron with respect to the ligands and to the heme plane. The anomalous behavior of cytochrome c oxidase, which exhibits only one EPR signal which is independent of temperature, suggests that the NO-oxygen may interact with the cuprous ion at the cytochrome as-Cu., site of the enzyme.