Effects of site-directed mutations in Escherichia coli succinate dehydrogenase on the enzyme activity and production of superoxide radicals.
Escherichia coli succinate dehydrogenase (SdhCDAB) catalyzes the oxidation of succinate to fumarate in the Krebs cycle, and during turnover, it produces superoxide radicals. SdhCDAB is a good model system for the succinate dehydrogenase (Sdh) found in the mitochondrial respiratory chain (complex II), as the subunits are structural homologues. Although mutations in sdh genes are reportedly associated with a variety of mitochondria-related diseases, the molecular mechanism of these diseases is poorly understood. We have investigated the effects of site-directed mutations around the heme (SdhD-H71L and SdhC-H91L), and at the ubiquinone-binding site (Q site; SdhC-I28E), on enzyme activity and production of superoxide radicals. The mutations SdhD-H71L and SdhC-I28E, but not SdhC-H91L, significantly reduce the succinate-ubiquinone reductase activity of the enzyme. All 3 mutant enzymes produce more superoxide than the wild-type enzyme, indicating that disturbance of the heme or the Q site can enhance superoxide production. The presence of a Q-site inhibitor reduces superoxide production significantly. Furthermore, the yield of superoxide is substrate dependent and increases with succinate concentration from 0.1 to 10 mmol/L. Our results indicate that, in SdhCDAB, the Q site with bound ubiquinone is an important source of superoxide radicals.
Zhao
Zhongwei
Z
Membrane Protein Research Group, Department of Biochemistry, 474 Medical Sciences Building, University of Alberta, Edmonton, AB T6G 2H7, Canada.
Rothery
Richard A
RA
Weiner
Joel H
JH
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010416:Pentachlorophenol / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
D013481:Superoxides; D014451:Ubiquinone; D010416:Pentachlorophenol; D013385:Succinate Dehydrogenase