The Fe2+-HiS(F8) Raman band shape of deoxymyoglobin reveals taxonomic conformational substates of the proximal linkage

The band shape of the Raman line attributed to the Fe2+-N-epsilon(His(F8)) stretching mode in deoxymyoglobin contains significant information on the n ature of the Fe-His proximal linkage. Raman lines appearing close to it, ho wever, obscure the true line profile. To isolate this from its accompanying lines we use its isotopic shift of similar to1 cm(-1) when Fe-56 in natura l-abundance deoxymyoglobin is substituted by Fe-54. This enables us to isol ate the true line shape. We have measured this line shape in sperm whale my oglobin dissolved in a 66% vol/vol glycerol/water solution for nine tempera tures from 10 K to 300 K. The nu (Fe-His) band shows a complex temperature- dependent profile, with a shoulder on its high-frequency wing, which become s more prominent with increasing temperature. Detailed analysis reveals tha t the band is composed of five distinct lines attributable to taxonomic con formational substates of the nu (Fe-His) linkage. These are in thermodynami c equilibrium above the glass transition temperature T-f but freeze in into the thermodynamic distribution at T-f for lower temperatures. Alternative models that try to explain the nu (Fe-His) band shape by either an anharmon ic coupling of the nu (Fe-His) to a low-frequency heme doming mode or by co nformational substates related to a Gaussian distribution of iron out-of-pl ane displacements are at variance with the distinct features observed exper imentally.