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 o f the high- and low-spin states of the active site of the substrate-fr ee form of cytochrome P450cam. The goal of these studies was to determ ine whether there is an underlying mechanism coupling the spin-state c hange itself with the observed shifts in the optical spectra, in the a bsence of any other changes in the heme unit. UV-visible spectroscopy, specifically, a small shift in the Soret band, is routinely used in e mpirical 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 me chanistic link between the two properties and because it is possible t hat the observed Soret band shifts are caused directly by the differen ces between the two heme proteins being compared without the intermedi acy of a spin-state change. Comparison of the calculated spectra of th e 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 co nsists primarily of porphyrin pi-pi transitions. These results establ ish 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 th e 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, c learly demonstrating an underlying physical basis for the observed cor relation between ferric heme spin state and measured spectral shifts a ssociated with (pi-->pi) heme transitions.