The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding.
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Yang X, Yu L, He D, Yu CA
The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding.
J Biol Chem. 1998 Nov 27;273(48):31916-23.
- PubMed ID
- 9822661 [ View in PubMed]
- Abstract
When purified ubiquinone (Q)-depleted succinate-ubiquinone reductase from Escherichia coli is photoaffinity-labeled with 3-azido-2-methyl-5-methoxy-[3H]6-geranyl-1,4-benzoquinone ([3H]azido-Q) followed by SDS-polyacrylamide gel electrophoresis, radioactivity is found in the SdhC subunit, indicating that this subunit is responsible for ubiquinone binding. An [3H]azido-Q-linked peptide, with a retention time of 61.7 min, is obtained by high performance liquid chromatography of the protease K digest of [3H]azido-Q-labeled SdhC obtained from preparative SDS-polyacrylamide gel electrophoresis on labeled reductase. The partial N-terminal amino acid sequence of this peptide is NH2-TIRFPITAIASILHRVS-, corresponding to residues 17-33. The ubiquinone-binding domain in the proposed structural model of SdhC, constructed based on the hydropathy plot of the deduced amino acid sequence of this protein, is located at the N-terminal end toward the transmembrane helix I. To identify amino acid residues responsible for ubiquinone binding, substitution mutations at the putative ubiquinone-binding region of SdhC were generated and characterized. E. coli NM256 lacking genomic succinate-Q reductase genes was constructed and used to harbor the mutated succinate-Q reductase genes in a low copy number pRKD418 plasmid. Substitution of serine 27 of SdhC with alanine, cysteine, or threonine or substitution of arginine 31 with alanine, lysine, or histidine yields cells unable to grow aerobically in minimum medium with succinate as carbon source. Furthermore, little succinate-ubiquinone reductase activity and [3H]azido-Q uptake are detected in succinate-ubiquinone reductases prepared from these mutant cells grown aerobically in LB medium. These results indicate that the hydroxyl group, the size of the amino acid side chain at position 27, and the guanidino group at position 31 of SdhC are critical for succinate-ubiquinone reductase activity, perhaps by formation of hydrogen bonds with carbonyl groups of the 1,4-benzoquinone ring of the quinone molecule. The hydroxyl group, but not the size of the amino acid side chain, at position 33 of SdhC is also important, because Ser-33 can be substituted with threonine but not with alanine.