Putidaredoxin Competitively Inhibits Cytochrome b₅-Cytochrome P-450_cam Association: A Proposed Molecular Model for a Cytochrome P-450_cam Electron-Transfer Complex

Cytochrome b₅ has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450_cam 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-450_cam 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-450_cam surface. Since the crystal structures for both cytochrome b₅ and cytochrome P-450_cam 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-450_cam 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-450_cam 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-450_cam electrostatic contacts.