THE QUINONE-BINDING SITE IN SUCCINATE-UBIQUINONE REDUCTASE FROM ESCHERICHIA-COLI - QUINONE-BINDING DOMAIN AND AMINO-ACID-RESIDUES INVOLVED IN QUINONE BINDING

When purified ubiquinone (Q)-depleted succinate-ubiquinone reductase f rom Escherichia coli is photoaffinity-labeled with 3-azido-2-methyl-5- methoxy-[H-3] 6-geranyl-1,4-benzoquinone ([H-3]azido-Q) followed by SD S-polyacrylamide gel electrophoresis, radioactivity is found in the Sd hC subunit, indicating that this subunit is responsible for ubiquinone binding, An [H-3]azido-Q-linked peptide, with a retention time of 61. 7 min, is obtained by high performance liquid chromatography of the pr otease It digest of [H-3]azido-Q-labeled SdhC obtained from preparativ e SDS-polyacrylamide gel electrophoresis on labeled reductase, The par tial N-terminal amino acid sequence of this peptide is NH2-TIRFPITAIAS ILHRVS-, corresponding to residues 17-33, The ubiquinone-binding domai n in the proposed structural model of SdhC, constructed based on the h ydropathy plot of the deduced amino acid sequence of this protein, is located at the N-terminal end toward the transmembrane helix I. To ide ntify amino acid residues responsible for ubiquinone binding, substitu tion 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 succin ate-Q reductase genes in a low copy number pRKD418 plasmid, Substituti on of serine 27 of SdhC with alanine, cysteine, or threonine or substi tution 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 an d [H-3]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 S dhC are critical for succinate-ubiquinone reductase activity, perhaps by formation of hydrogen bonds with carbonyl groups of the 1,4-benzoqu inone ring of the quinone molecule. The hydroxyl group, but not the si ze of the amino acid side chain, at position 33 of SdhC is also import ant, because Ser-33 can be substituted with threonine but not with ala nine.