Resonance Raman structural characterization of cyanide ligated cytochrome P450cam
Abstract
Cytochromes P450 are the strongest known in vivo oxidizing agents whose essential function is the oxidative metabolism of a large variety of compounds. The essential goal of the present work is to utilize resonance Raman (RR) spectroscopy to study the active site structure of cyanide ligated cytochrome P450cam as an analogue to the ferric-superoxo P450cam, an intermediate involved in the oxidation of the substrate. It has been recently proposed that the key factor in catalyzing the cleavage of the bound O$\sb2$ is the involvement of an active site hydrogen bond donor. In order to investigate this issue. RR studies of the enzyme-cyanide complexes of the wild-type enzyme were performed. Careful analyses of the difference patterns obtained by subtraction of various pairs of spectra of four CN$\sp-$ isotopomers provide convincing evidence for the presence of two structural conformers; one "essentially linear" and the other bent. Both conformers persist for the substrate-bound derivatives. The linear conformer exhibits the $\nu$(Fe-C) stretching mode at 413 cm$\sp{-1}$ and the $\delta$(FeCN) bending mode at 387 cm$\sp{-1}$ for the substrate-free derivative. The corresponding values for the camphor-bound form are 416 and 392 cm$\sp{-1}$, while for the adamantanone derivative these occur at 423 and 387 cm$\sp{-1}$. The bent conformer exhibits a set of vibrational parameters which is characterized by a "zig-zag" isotope shift pattern for both the lower frequency and the higher frequency mode. For the substrate-free form, the vibrational modes of both conformers are shown to be sensitive to $\sp1$H$\sb2$O/$\sp2$H$\sb2$O exchange, confirming that they are both hydrogen bonded. While in the case of the linear conformer the H-bond donor is most likely the active site water cluster, the (presumably off-axis) donor for the bent conformer may be either another region of the water cluster or the threonine-252 (or aspartate-251) residue which may be in a position to interact with the polar CN$\sp-$ ligand. The vibrational frequencies of both conformers are sensitive to substrate binding and to the substrate size. Furthermore, the vibrational modes of both conformers are insensitive to $\sp1$H$\sb2$O/$\sp2$H$\sb2$O exchange in the substrate-bound forms. While the lack of $\sp1$H$\sb2$O/$\sp2$H$\sb2$O sensitivity does not exclude the possibility that one or both conformers are H-bounded, its absence, together with the demonstrated sensitivity to substrate size, suggests that steric factors are important in determining the geometry of the FeCN fragment. Finally, all the experimentally derived frequencies and isotopic shifts are shown to be consistent with the predictions of normal mode calculations for these two conformers.
This paper has been withdrawn.