DETERMINANTS OF THE VINYL STRETCHING FREQUENCY IN PROTOPORPHYRINS - IMPLICATIONS FOR COFACTOR-PROTEIN INTERACTIONS IN HEME-PROTEINS

Soret-excitation resonance Raman (RR) spectra are reported for the bis-imidazole complexes of a series of mono- and divinylhemins. The complexes include 2-vinyldeuterohemin IX, 4-vinyldeuterohemin IX, protohemin IX, and protohemin III. For all four hemins, two polarized RR bands are observed at similar to 1620 and similar to 1631 cm(-1). Both of these bands are absent from the spectrum of the deuterohemin IX, which contains no vinyl substitutents, The relative intensities of the 1620- and 1631-cm(-1) bands are similar to 60:40 for all of the vinylhemins studied. However, the intensity of each band of both monovinyl complexes is approximately one-half that of the analogous bands of both divinyl complexes. The appearance of the 1620- and 1631-cm(-1) bands is independent of solvent although the 1631-cm(-1) band is difficult to identify in aqueous solutions wherein the hemins are aggregated. Temperature-dependent RR studies indicate that the intensity of the 1630-cm(-1) band monotonically decreases relative to that of the 1620-cm(-1) feature as the temperature is lowered. The 1620-cm(-1) feature has generally been assigned as the characteristic vinyl stretching mode (nu(C=C)) of vinylhemins. The 1631-cm(-1) band has not been previously identified in the RR spectra of vinylhemins in solution but has been observed in the spectra of heme proteins which contain protohemin M. For the proteins, the 1631-cm(-1) band has been assigned as a second nu(C=C) mode. The appearance of two nu(C=C) modes has generally been attributed to site-specific vinyl group-protein interactions which render the 2- and 4-vinyl substituents of the protohemin IX cofactor inequivalent. In the case of the vinylhemins in solution, we also assign the 1631-cm(-1) band to a second nu(C=C) mode. However,the simultaneous appearance of two nu(C=C) modes is attributed to the existence of two nearly equal-energy vinyl torsional conformers which are intrinsic to a single vinylgroup. In the divinyl complexes, both conformers occur for each vinyl group; however, the 2- versus 4-vinyl substituents cannot be distinguished due to the absence of vibrational coupling, Local density functional calculations on a vinylporphyrin model and several vinyl-substituted small molecules confirm that two vinyl torsional conformers should exist and that these conformers are close in energy (within 450 cm(-1) or less). In the porphyrin, the vinyl group of the lower energy form (Conformer I) is nearly in plane and points toward the beta-pyrrole methyl group. The vinyl group of the higher energy form (Conformer II) is out of plane by similar to 40 degrees and points toward the meso-hydrogen. Explicit second-derivative calculations on the small molecules indicate that the frequencies of the nu(C=C) modes of the two vinyl torsional conformers differ by 10-20 cm(-1). The calculations further suggest that the 1620- and 1631-cm(-1) nu(C=C) modes observed for the vinylhemins in solution are associated with Conformers I and II, respectively. The fact that vinylhemins can occupy two nearly-equal energy torsional conformers has significant implications for the interpretation of the RR spectra of proteins that contain protohemin IX. In particular, the appearance of two nu(C=C) modes does not necessarily justify an interpretation which invokes site-specific vinyl group-protein interactions.