Residue 285 in cytochrome P450 2B4 lacking the NH(2)-terminal hydrophobic sequence has a role in the functional association of NADPH-cytochrome P450 reductase.
Cytochrome P450 2B4 (CYP2B4) lacking the NH(2)-terminal signal anchor sequence (2-27) was used to study the impact of replacement of histidine with alanine at position 285 on electron transfer from NADPH-cytochrome P450 reductase (P450R). Absorption and circular dichroism spectra of the recombinant hemoproteins indicated that amino acid substitution neither grossly perturbed the geometry of the immediate heme vicinity nor the global polypeptide backbone folding. Fitting of the initial-velocity patterns of P450R-directed reduction 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 association of the redox partners accounted for this phenomenon. Thus, deletion of the positive charge at position 285 of CYP2B4 (2-27) might have disrupted contacts with oppositely charged entities on the P450R surface. Measurements of the stoichiometry of aerobic NADPH consumption and H(2)O(2) 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 truncated CYP2B4 is of importance in the functional interaction with the flavoprotein reductase.
Schulze
J
J
Walther-Straub-Institut für Pharmakologie und Toxikologie der LMU, Nussbaumstrasse 26, Munich, D-80336, Germany.
Tschöp
K
K
Lehnerer
M
M
Hlavica
P
P
D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase