Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex.
Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.
Stayton
P S
PS
Department of Biochemistry University of Illinois, Urbana 61801.
Poulos
T L
TL
Sligar
S G
SG
D001667:Binding, Competitive; D019475:Camphor 5-Monooxygenase; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015786:Cytochromes b5 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology; D005288:Ferredoxins / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008956:Models, Chemical; D009154:Mutation; D011487:Protein Conformation
D065607:Cytochrome P-450 Enzyme Inhibitors; D005288:Ferredoxins; D005456:Fluorescent Dyes; C100137:putidaredoxin; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase