SITE-DIRECTED MUTAGENESIS OF THE CONSERVED THREONINE (THR243) OF THE DISTAL HELIX OF FUNGAL CYTOCHROME P450NOR

Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduc tion in denitrifying fungi. Threonine 243 (Thr243) of P450nor, which c orresponds to the conserved threonine of monooxygenase cytochrome P450 s, was replaced by 18 different amino acids via site-directed mutagene sis. The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligatio n, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural change s in the heme environment on Thr243 mutation. However, the NO reductio n activity was dramatically altered by Thr243 mutation, depending on t he properties of the replaced amino acids. The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retai ned on substitution of Asn, Ser, and Gly for Thr243, while it was prof oundly decreased or lost on substitution with other amino acids. Kinet ic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainl y results from a decrease in the rate of reduction of the ferric-NO co mplex with NADH, On the basis of these enzymatic, kinetic, and spectro scopic results, as well as on the basis of the crystal data for native P450nor [Park, S.-Y., et al. (1997) Not. Struct. Biol, 4, 827-832], t he role of the conserved threonine at the 243 position in the NO reduc tion reaction by P450nor is discussed. We also discuss structural simi larities or differences in the vicinity of the conserved threonine bet ween P450nor and other monooxygenase P450s.