The Cytochrome P-450_cam Binding Surface As Defined by Site-Directed Mutagenesis and Electrostatic Modeling

Cytochrome P-450_cam cationic surface charges at Lys 344, Arg 72, and Lys 392 have been altered by site-directed mutagenesis techniques. The residues at Lys 344 and Arg 72 were previously suggested as salt bridge contacts in the cytochrome b₅-cytochrome P-450_cam association complex and implicated in the physiological putidaredoxin-cytochrome P-450_cam complex [Stayton, P. S., Poulos, T. L., & Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. Mutations to neutralize the basic charge at Arg 72 (R72Q) and to both neutralize and reverse the charge at Lys 344 (K344Q, K344E) resulted in alteration of NADH oxidation rates in the reconstituted physiological electron-transfer system, which is rate limited by putidaredoxin-cytochrome P-450_cam electron transfer. The steady-state V_max values were apparently unperturbed, suggesting that the observed rate differences were largely attributable to K_m effects. The K_m values observed for the K344Q (24 μM) and K344E (32 μM) mutants are in the direction expected for neutralization and reversal of a salt bridge charge interaction. A control mutation at a basic surface charge located away from the proposed site of interaction, Lys 392 (K392Q), resulted in overall activities quantitated by NADH oxidation rates that are similar to that of wild-type cytochrome P-450_cam. Calculation of the cytochrome P-450_cam electrostatic field revealed a patch of positive potential at the modeled cytochrome b₅ interaction site lying directly above the nearest proximal approach to the buried heme prosthetic group. These results provide experimental and theoretical evidence for the modeled cytochrome P-450_cam binding site implicated in both cytochrome b₅ and putidaredoxin association. These results also provide an interesting framework for consideration of how well eukaryotic sequences and functional studies correlate with the cytochrome P-450_cam structural model.