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 compl exes include 2-vinyldeuterohemin IX, 4-vinyldeuterohemin IX, protohemi n 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 th ese bands are absent from the spectrum of the deuterohemin IX, which c ontains 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 complex es is approximately one-half that of the analogous bands of both divin yl complexes. The appearance of the 1620- and 1631-cm(-1) bands is ind ependent of solvent although the 1631-cm(-1) band is difficult to iden tify in aqueous solutions wherein the hemins are aggregated. Temperatu re-dependent RR studies indicate that the intensity of the 1630-cm(-1) band monotonically decreases relative to that of the 1620-cm(-1) feat ure as the temperature is lowered. The 1620-cm(-1) feature has general ly been assigned as the characteristic vinyl stretching mode (nu(C=C)) of vinylhemins. The 1631-cm(-1) band has not been previously identifi ed in the RR spectra of vinylhemins in solution but has been observed in the spectra of heme proteins which contain protohemin M. For the pr oteins, the 1631-cm(-1) band has been assigned as a second nu(C=C) mod e. The appearance of two nu(C=C) modes has generally been attributed t o site-specific vinyl group-protein interactions which render the 2- a nd 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 equa l-energy vinyl torsional conformers which are intrinsic to a single vi nylgroup. In the divinyl complexes, both conformers occur for each vin yl group; however, the 2- versus 4-vinyl substituents cannot be distin guished due to the absence of vibrational coupling, Local density func tional calculations on a vinylporphyrin model and several vinyl-substi tuted small molecules confirm that two vinyl torsional conformers shou ld 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 fo rm (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 molecul es 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, resp ectively. The fact that vinylhemins can occupy two nearly-equal energy torsional conformers has significant implications for the interpretat ion of the RR spectra of proteins that contain protohemin IX. In parti cular, the appearance of two nu(C=C) modes does not necessarily justif y an interpretation which invokes site-specific vinyl group-protein in teractions.