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 analog...
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Published in | Biochemistry (Easton) Vol. 39; no. 44; pp. 13496 - 13502 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
07.11.2000
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ark:/67375/TPS-VHWRQRCG-5 istex:44582CB5541EF295E005D17FA72DE6EF8A4BAA7D This work was supported by the Academy of Finland [Grant 43660, Cell Biology Program, and Program 44895, Structures and Catalytic Mechanism of Membrane Proteins (Finnish Center of Excellence Programme 2000−2005)] and the Sigrid Juselius Foundation. M.F. is indebted to EMBO for a short-term fellowship, and V.Z. is indebted to the Deutsche Forschungsgemeinschaft (DFG) for financial support. ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi001134s |