Identification of key residues in rabbit liver microsomal cytochrome P4502B4: Importance in interactions with NADPH-cytochrome P450 reductase

A cytochrome P450 2B4 (CYP2B4) model was used to select key residues suppos ed to serve in interactions with NADPH-cytochrome P450 reductase (P450R), E ight amino acid residues located on the surface of the hemoprotein were cho sen for mutagenesis experiments with CYP2B4(Delta 2-27) lacking the NH2-ter minal signal anchor sequence. The mutated proteins were expressed in Escher ichia coli, purified, and characterized by EPR- and CD-spectral analysis. R eplacement of histidine 226 with alanine caused a 3.8-fold fall in the affi nity for P450R with undisturbed reductive capacity of the system. Similarly , the K225A, R232A, and R253A variants exhibited P450R-directed activity th at was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta 2-27) resul ted in impaired electrostatic contacts with complementary amino acids on th e P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the pheny lalanine residues at positions 223 and 227 gave a 2.1- to 6.1-fold increase in the K-m values with unchanged V-max; this was attributed to the disrupt ion of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NA DPH consumption and H2O2 formation revealed the oxyferrous forms of the F22 3A, H226A, and F227A mutants to autoxidize more readily owing to less effic ient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-29 6 located close to the heme unit. All of the residues examined reside in th e putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.