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 (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.