On the stoichiometry of the iron‐sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase

Abstract

The concentration of the iron‐sulphur (Fe‐S) cluster 1b, present in complex I or soluble high‐molecular‐mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation‐broadened signals of the Fe‐S clusters 2–4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady‐state kinetic and inhibitor‐titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152–159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer. Copyright © 1992, Wiley Blackwell. All rights reserved