The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition

Abstract

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol-labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Cal-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein of trypsin indigestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the axido-Q-labeled peptide is located in the membrane on the matrix side. These results confirms our previous assessment that the Mr = 13400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment. Antibodies against QPc-9.5 kDa cause an increase of the apparent Km for ubiquinol 2 in ubiquinol-cytochome c reductase, suggesting that the low level of inhibition of the reductase by these antibodies may be due to the use of excess ubiquinol 2 in the assay mixture. Since antibodies against QPc-9.5 kDa inhibit 75% of the antimycin-sensitive plastoquinone reduction activity in the reconstituted succinate-cytochrome c reductase, QPc-9.5 kDa may be involved in the Qi site. The topological arrangement of QPc-9.5 kDa in the mitochrondrial membrane was examined immunologically with an anti-QPc-9.5 kDa Fab'' fragment-horseradish peroxidase conjugate. When intact mitochondria (mitoplasts) or electron-transport particles (ETP) are exposed to this conjugate, peroxidase activity is found in both preparations, with ETP having the higher activity. This suggests that QPc-9.5 kDa is transmembranous, possibly with more mass on the matrix side of the membrane.