Heme proteins that have been reconstituted with certain hemins may contain substantial fractions of a minor component in which the orientation of the heme in the folded pocket differs from the major (“native”) conformation by a 180° rotation about the α-γ meso axis. In fact, this minor component has also been shown to exist in some native proteins, including several mammalian globins. While resonance Raman spectroscopy has emerged as a powerful probe of active site structure of heme proteins, no systematic study has yet been undertaken to elucidate the specific spectral changes associated with this disorder. In the present work, combined analyses of the temporal behavior of both NMR and RR data sets have been completed to permit the extraction of a unique RR spectrum for the disoriented form, documenting rather dramatic changes associated with this rotational disorder. In addition, the use of protohemes bearing selectively deuterated peripheral methyl groups has permitted the association of the observed modes with specific fragments of the heme residing in the reversed orientation. The studies conducted here clearly illustrate the exquisite sensitivity of low frequency heme deformation modes to altered protein-heme interactions. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 179–186, 2008.
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