MECHANISTIC ORIGIN OF THE CORRELATION BETWEEN SPIN-STATE AND SPECTRA OF MODEL CYTOCHROME-P450 FERRIC HEME-PROTEINS

We report here the use of the semiempirical quantum chemical INDO/ROHF/CI method to calculate the electronic structure and optical spectra of the high- and low-spin states of the active site of the substrate-free form of cytochrome P450cam. The goal of these studies was to determine whether there is an underlying mechanism coupling the spin-state change itself with the observed shifts in the optical spectra, in the absence of any other changes in the heme unit. UV-visible spectroscopy, specifically, a small shift in the Soret band, is routinely used in empirical correlations to determine the variation in percent high-spin and low-spin forms as a function of a specific change in heme proteins, for example, in the axial ligands, in the substrate binding, or in a mutant. These correlations are weakened, however, by the lack of a mechanistic link between the two properties and because it is possible that the observed Soret band shifts are caused directly by the differences between the two heme proteins being compared without the intermediacy of a spin-state change. Comparison of the calculated spectra of the two spin states reveals that, upon change of the Fe(III) from a low- to a high-spin reference state, a blue shift does indeed occur in the strong Soret band absorptions found in the 25 000 cm-1 region that consists primarily of porphyrin pi-pi* transitions. These results establish that a spin-state change alone leads to the observed shift in the Soret band. They also elucidate the origin of the observed shift in the Soret band and show it to be a direct consequence of the spin-state change. In the low-spin state, there is enhanced mixing of the iron d (eg) and porphyrin 4eg (pi*) orbitals, resulting in a lowering of the eg(pi*) states, and a consequent shift to the red of the Soret band, clearly demonstrating an underlying physical basis for the observed correlation between ferric heme spin state and measured spectral shifts associated with (pi-->pi*) heme transitions.