Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide.
Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.
Kurki
S
S
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, Helsinki, Finland.
Zickermann
V
V
Kervinen
M
M
Hassinen
I
I
Finel
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D017124:Conserved Sequence / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D018698:Glutamic Acid / Q000235:genetics*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014451:Ubiquinone / Q000378:metabolism*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D014451:Ubiquinone; D018698:Glutamic Acid; D004024:Dicyclohexylcarbodiimide; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I