Mutation effects of a conserved threonine (Thr243) of cytochrome P450nor on its structure and function

Threonine 243 of cytochrome P450nor (fungal nitric oxide reductase) corresp onds to the 'conserved' Thr in the long I helix of monooxygenase cytochrome P450s. In P450nor, the replacement of Thr243 with Asn, Ala or Val makes th e enzymatic activity dramatically reduce. In order to understand the roles of Thr243 in the reduction reaction of NO by P450nor, the crystal structure s of three Thr243 mutants (Thr243-->Asn, Thr243-->Val, Thr243-->Ala) of P45 0nor were determined at a 1.4-Angstrom resolution and at cryogenic temperat ure. However, the hydrogen-bonding pattern in the heme pocket of these muta nts is essentially similar for that of the WT enzyme. This suggests that th e determination of the structure of the NADH complex of P450nor is required , in order to evaluate the role of Thr243 in its enzymatic reaction. We att empted to crystallize the NADH complex under several conditions, but have n ot yet been successful. (C) 2000 Elsevier Science BN. All rights reserved.