Reconstitution of the Ubiquinol: Cytochrome c Reductase from a bc1 Subcomplex and the 'Rieske' Iron-Sulfur Protein Isolated by a New Method

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Abstract
A method for preparing the Rieske Fe-S protein and the bc1 subcomplex of complex III was developed. The new method is advantageous over the published ones in that: the final yield and amount exceeds by far those obtained when employing the hitherto published methods; Fe-S protein as well as the bc1 subcomplex are obtained by one and the same preparation procedure from a common source; and the preparation method is easier than the published ones. The Fe-S protein obtained represents the first reconstitutively active preparation present in a monodisperse state. The reconstitution of the ubiquinol:cytochrome c reductase from the 2 components is a reversible dissociation process. Full activity of ubiquinol:cytochrome c reductase is reached after saturation of the binding site of the bc1 subcomplex for Fe-S protein. Full reduction of the constituent cytochrome c1 of bc1 subcomplex can already be obtained with substoichiometric amounts of Fe-S protein, however. The question might be raised whether the observed dissociation equilibrium represents merely a phenomenon occurring specifically with the proteins isolated in Triton X-100 and investigated in a Triton-containing buffer, or whether dissociation of the Fe-S protein also takes place in the mitochondrial membrane in the course of the electron-transfer reaction sequence.

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