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
S. Kurki et al., 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, BIOCHEM, 39(44), 2000, pp. 13496-13502
Steady-state kinetics of the H+-translocating NADH:ubiquinone reductase (co
mplex 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 analo
gues were in the millimolar range. Both the structure and size of the hydro
phobic side chain of the acceptor affected its affinity fur complex I. The
ND1 subunit of bovine complex I is a mitochondrially encoded protein that b
inds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hat
efi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. debitri
ficans complex I is a homologue of ND1, and within it three conserved Glu r
esidues that could bind DCCD, E158, E212, and E247, were changed to either
Asp or Gin 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 ubiquinon
es, The implications of the results for the location of the ubiquinone redu
ction site in this enzyme are discussed.