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Resonance Raman determination of vinyl group disposition in different derivatives of native myoglobin and its heme-disoriented form



In heme-reconstituted heme proteins, the heme inserts such that some of the heme is rotated 180° about the α–γ meso axis, eventually equilibrating to native conformations. Also, proteins from different species may naturally possess both conformers. Resonance Raman (RR) spectroscopy is effective in signaling this conformational heterogeneity, detecting altered interactions with active site residues. While shifts of the vibrational modes of the two vinyl groups could be readily detected, they could not be assigned to a particular vinyl group. This deficiency is alleviated by employing a specifically labeled protoheme isotopomer, wherein only the 4-vinyl group is labeled (i.e. –CH=C2H2), providing a spectral editing mechanism for selectively tracking the individual vinyl groups. The RR spectral data acquired here for the met-, deoxy-, and ferrous CO adduct of myoglobin, along with their ‘flipped heme’ isomers, provide convincing evidence for the anticipated effects of ‘swapping’ the environments of the two vinyl groups; i.e., in the native form, the 2-vinyl group assumes a more out-of-plane orientation with respect to the pyrrole plane than does the (nearly in-plane) 4-vinyl group, whereas in the ‘reversed’ orientation, the 4-vinyl group now assumes a position that is more out-of-plane than the newly positioned 2-vinyl group. The ability of RR spectroscopy to document such differences in the orientation of the vinyl substituents is made important by the fact that changes in the disposition of these groups has long been known to carry functional consequences. Copyright © 2014 John Wiley & Sons, Ltd.