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 b 5 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