Resonance Raman and EPR investigations of the D251N oxycytochrome p450(cam)/putidaredoxin complex

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450 (cam) (oxy-P450(cam)) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450(cam)/Pd complex has a perturbed proton delivery mechanism an d shows a significantly red-shifted UV-visible spectrum as observed in Bens on et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemi stry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the hem e active site of D251N oxy-P450(cam). This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processe s may be coupled. Three oxygen isotope sensitive Raman features are identif ied in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam How ever, a careful examination of the oxygen stretching feature near 1137 cm(- 1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1) which we attribute to the presence of conformational substates in oxy-P450(cam). A significant change in the conformational substate population is observed for the D251N oxy-P450(cam) when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450(cam), along with the red-shifted electronic spectra, reflects a structural equilibrium of th e oxy-heme that is perturbed upon Pd binding. We propose that this structur al perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.