Interaction between mitochondrial CYP27B1 and adrenodoxin: Role of arginine 458 of mouse CYP27B1

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K-m values for 1 alpha-hydroxylation of 25(OH)D-3 indicate that the replacement of Arg458 with Lys or Gin does not affect substrate binding. However, these mutants showed remarkable decreases of both k(cat) values and the ratio of product fori-nation to NADPH oxidation (coupling efficiency). A high K-m value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gin abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1 alpha-hydroxylation of 25(OH)D-3. On the other hand, binding one ADX molecule is sufficient for the conformational chattge of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.