CHARACTERIZATION OF THE ROLE OF LYSINE 110 OF NADH-CYTOCHROME-B5 REDUCTASE IN THE BINDING AND OXIDATION OF NADH BY SITE-DIRECTED MUTAGENESIS

An expression vector for bovine NADH-cytochrome b5 reductase was used for site-directed mutagenesis of lysine 110, the residue previously implicated in NADH interactions with this flavoprotein. Replacement of this basic residue with an uncharged glutamine resulted in an increase of 3 orders of magnitude in the K(m) for NADH and a decrease in k(cat) of an order of magnitude, strongly implicating lysine 110 in both binding of NADH to the reductase and the orientation of the reduced nicotinamide group for rapid hydride ion transfer to the flavin. Substitution of lysine 110 by histidine, to provide a pH-sensitive positive charge at this position in the neutral pH range, exhibited only a moderate 25-fold increase in K(m) and a normal k(cat) at pH 6.0, whereas at pH 8.5 the K(m) for NADH rose to 238 muM with a decrease of 45% over unmodified enzyme in the k(cat). A similar pH sensitivity in the inhibition constant for adenosine diphosphate ribose, lacking only the nicotinamide moiety of NADH, emphasizes the crucial role of the positive charge at this locus and is consistent with charge-pairing of lysine 110 with the pyrophosphate group of NADH or adenosine diphosphate ribose.