Computation of Transient Intermediate Spectra:

P450 Compound I

Reliable computation of spectral shifts of transient intermediate of heme proteins may be obtained at a semiempirical INDO/ROHF/CI level of theory, given accurate experimental or computed geometries. Such an approach is useful because it can provide quantitative estimates of the spectral relationship between transient intermediates and known stable species. While the absolute positions of spectral transitions cannot, in general, be obtained at a CI-singles (CIS) level of theory, computed spectral shifts between species are quantitive. The results of such an approach permits one to make species-transient spectra associations.

The optimized structure of model P450 compound I below was computed using nonlocal density functional theory (DFT) employing a BPW91 functional and a DZVP basis set. While our current DFT work, investigating reaction pathways, employs more accurate hybrid functionals, spectra computed at BPW91 geometries are adequate for this purpose. Shown in Panel A below are the computed spectra of compound I of P450s compared with the computed resting state spectrum. Analogous to the experimental spectrum shown below it in Panel B), the Soret maximum of model P450 compound I is ca. 60 nm blue shifted with respect to the resting state maximum. The spectrum was computed at the optimized DFT geometry. This work was recently reported in the Journal of Inorganic Biochemistry (Harris, Loew, and Waskell 83:309-318, 2001).


Computed (A) and Experimental (B) Compound I and Resting State P450 Heme Species Spectra.

Oxyferrous and Reduced Oxyferrous P450 Heme Species.

Benson, Suslick and Sligar (Biochemistry, 1997, 36, 5104) determined the spectrum of a transient species which resulted from the irradiation of the oxyferrous D251N mutant P450cam. The spectrum of the transient species was red-shifted by ca. 30 nm. In a DFT and INDO/CI study of these species we computed the ground state geometries and properties of the oxyferrous and reduced oxyferrous species(Harris, Loew, and Waskell, 120:4308-318 1998). Both the oxyferrous and reduced oxyferrous heme species were found to be end-on O2 bound structures. The structure of the reduced oxyferrous heme species is shown below:

Shown below is the reduced oxyferrous heme spectra computed at DFT optimized doublet ground state. Shown as an inset to this figure is the experimental spectrum of Benson and Sligar.