A Study Of The Resonance Raman Spectra Of Some Heme Proteins And Other Molecules PDF Download

One effective approach for exploring structure/function relationships in heme proteins is to study proteins that have been reconstituted with modified hemes so as to systematically perturb the protein-heme interface. However, some reconstituted heme proteins may contain substantial fractions of a "non native" state in which the orientation of the heme in the folded pocket differs from the native conformation by a 180° rotation about the [alph alpha - gamma gamma] meso axis. In fact, this "non native" state has also been shown to exist in some native proteins, including several mammalian globins. In order to define changes in the active site structure associated with this "disorder", we have applied resonance Raman spectroscopy to the metMb derivatives, using selectively deuterated protohemes to associate the observed modes with specific fragments of the heme. Resonance Raman spectroscopy is also employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives; i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe-CO and Fe-CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the [upsilon](Fe-NHis), [upsilon](Fe-C) and [upsilon](C-O) modes. We have synthesized protohemes with selectively labeled vinyl groups and have effectively reconstituted them into apo-myoglobin in order to assign the so-called "vinyl bending" modes of heme group in native and reversed forms of myoglobin to their specific molecular fragments based on their isotopic shift with these vinyl labeled protohemes. In a separate project, these vinyl labeled hemes have been employed to further define structural changes in cytochrome P450cam upon substrate binding. Substrate binding to cytochrome P450cam is known to induce the distortions of the out of plane modes such as [gamma gamma]6 and [gamma gamma]7 modes as well as the heme peripheral substituents. The detection of these low frequency modes is especially important as the disposition of these groups can modify the heme reduction potential.