Role of Lys-110 of human NADH-cytochrome b5 reductase in NADH binding as probed by site-directed mutagenesis.

Lys-110 of human NADH-cytochrome b5 reductase was replaced by Ala, Met, or Arg by site-directed mutagenesis to evaluate the role of the residue. Km values of purified Lys-110-->Ala and Lys-110-->Met mutants for NADH were approximately 200-fold and 1,100-fold higher than that of the wild-type, respectively, while the value of the Arg mutant was almost the same as that of the wild-type. These results indicate that the positive charge at position 110 is important for NADH binding. The kcat value of Lys-110-->Ala was not affected, indicating that the residue only participates in the binding process in the reaction by forming an ionic interaction with phosphoryl group of NADH.