Mitochondrial ubiquinol-cytochromec reductase complex: Crystallization and protein: Ubiquinone interaction

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Abstract
The ubiquinol-cytochromec reductase complex was crystallized in a thin plate form, which diffracts X-rays to 7 Å resolution in the presence of mother liquor. This crystalline complex contains ten protein subunits and 140 nmol phospholipid per milligram protein. Over 90% of the phospholipid and ubiquinone in the reductase can be removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The delipidated complex has no enzymatic activity and shows significant changes in the circular dichroism spectrum in the near UV region and in the EPR characteristics of both cytochromesb. Enzyme activity and spectral characteristics can be restored by replenishing the phospholipid and ubiquinone. The structural requirements of ubiquinone for electron transport were studied by measuring the ability of a variety of synthetic ubiquinone derivatives to restore the enzymatic activity and native spectroscopic signatures to the delipidated complex. Q-binding proteins and binding domains were identified using photoaffinity labeled Q-derivatives and HPLC separation of photolabeled peptides. Interaction between ubiquinol-cytochromec reductase and succinate-Q reductase was established by differential scanning calorimetry and saturation transfer EPR using spinlabeled ubiquinol-cytochromec reductase. Involvement of iron-sulfur protein in proton translocation by ubiquinol-cytochromec reductase was investigated by hematorporphyrinpromoted photoinactivation of the complex. ThecDNAs encoding the Rieske iron-sulfur protein and a small molecular mass Q-binding protein (QPc-9.5 kDa) were isolated and their nucleotide sequences determined. These will be useful in future structural and mechanistic studies of ubiquinol-cytochromec reductase viain vitro reconstitution between an overexpressed, mutated subunit and a specific subunit-depleted reductase.

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