Selective removal of spectral components in complex ST-EPR spectra of spin-labeled cytochrome P-450

Abstract

Incubation of cytochrome P-450 spin labeled by an isocyanide derivative with K 3Fe(CN) 6 results in a selective disappearance of the spectral components attributable to the weakly immobilized spin labels in the conventional EPR and ST-EPR spectra. The lineshape of that part of the spin-label spectrum which corresponds to the strongly immobilized spin-label molecules remains unaltered. This selective disappearance of distinct parts in complex spectra is important for the analysis and interpretation of ST-EPR spectra from which rotational correlation times are derived. Its applicability is demonstrated for the hepatic membrane-bound cytochrome P-450 of the endoplasmic reticulum, for which the mobility of the spin-labeled isocyanide derivative bound to the active site of the enzyme was determined.