ESEEM and ENDOR studies of the Rieske iron-sulphur protein in bovine heart mitochondrial membranes

Abstract

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) were applied to the respiratory-chain iron-sulphur clusters in natural bovine heart mitochondrial membranes. By using specific reduction, signals were observed from the Complex III Rieske [2Fe-2S] cluster. In ENDOR, 1H hyperfine couplings in the range 0.5–7 MHz were observed. In ESEEM, modulations were obtained which were assigned to two 14N nuclei of directly-coupled imidazole ligands. The ESEEM spectra are similar to previous observations on purified iron-sulphur proteins of this type, in which the iron-sulphur cluster is coordinated by two cysteine and two histidine ligands. They confirm that the coordination of the cluster in the purified proteins, with two cysteinyl sulphur and two histidine nitrogens, is unchanged from its natural mitochondrial membrane environment. In order to investigate the possible interaction of the membrane-bound Rieske protein with quinones, measurements were conducted on membranes preincubated with 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), and in the pH range 6–7.5. No significant changes were detected, either in the proton hyperfine couplings as detected by ENDOR, or in the nitrogen couplings to the histidines as detected by ESEEM.