Residue 285 in cytochrome P4502B4 lacking the NH2-terminal hydrophobic sequence has a role in the functional association of NADPH-cytochrome P450 reductase

Cytochrome P450 2B4 (CYP2B4) lacking the NH2-terminal signal anchor sequenc e (2-27) was used to study the impact of replacement of histidine with alan ine at position 285 on electron transfer from NADPH-cytochrome P450 reducta se (P450R). Absorption and circular dichroism spectra of the recombinant he moproteins indicated that amino acid substitution neither grossly perturbed the geometry of the immediate heme vicinity nor the global polypeptide bac kbone folding. Fitting of the initial-velocity patterns of P450R-directed r eduction of the ferric CYP2B4 (2-27) forms to the Michaelis-Menten kinetics revealed an approximately 3.5-fold increase in the apparent K-m value for the electron donor of the H285A mutant, while its reductive capacity (V-max ) remained unchanged; this caused a strong drop in reductive efficiency of the engineered enzyme, Circumstantial analysis suggested that impaired asso ciation of the redox partners accounted for this phenomenon. Thus, deletion of the positive charge at position 285 of CYP2B4 (2-27) might have disrupt ed contacts with oppositely charged entities on the P450R surface, Measurem ents of the stoichiometry of aerobic NADPH consumption and H2O2 production disclosed the oxyferrous H285A species to autoxidize more readily compared with the shortened wild type. This was assumed to arise from less efficient coupling of the system due to defective donation of the second electron by P450R. These results are consistent with the view that His-285 in the trun cated CYP2B4 is of importance in the functional interaction with the flavop rotein reductase. (C) 2000 Academic Press.