PROTEIN-HEME INTERACTIONS IN HEMOGLOBIN FROM THE MOLLUSK SCAPHARCA-INAEQUIVALVIS - EVIDENCE FROM RESONANCE RAMAN-SCATTERING

Resonance Raman spectra of the Scapharca inaequivalvis homodimeric hem oglobin (HbI) have been measured for the ligand-bound and ligand-free ferrous forms of the protein. In the deoxy derivative, the iron-histid ine (Fe-His) stretching mode, proposed as a marker of the oxygen affin ity and a conduit linking the hemes to the subunit interface, gives ri se to a Raman peak centered at 203 cm-1, an unusually low frequency co mpared to that reported for other hemoglobins and myoglobins. In the C O-bound derivative, three isotope-sensitive lines at 517, 583, and 194 5 cm-1 have been assigned to the Fe-CO stretching, Fe-C-O bending, and C-O stretching modes, respectively. From the frequencies of these mod es and from their relative intensities, the Fe-C-O geometry appears to be tilted from axial coordination and shows a bending angle which has been estimated to be about 171 +/- 5-degrees. For the oxygen derivati ve, only one isotope-sensitive peak has been detected at 570 cm-1, in line with the values found for myoglobin and other hemoglobins. Resona nce Raman spectra of HbI modified with p-(chloromercuri)benzoate (PMB) at Cys92 have been measured in parallel with those of the native prot ein. Despite the large increase in oxygen affinity produced by the PMB modification, the frequency of the Fe-His stretching mode is unshifte d in the deoxy derivative. Thus, in HbI, the frequency of the Fe-His s tretching mode does not correlate with the dissociation constant of th e last oxygen molecule as found in human hemoglobins. In turn, this fi nding indicates that the iron-histidine bond does not play the same ro le in heme-heme communication in HbI as it does in the vertebrate hemo globins. The low-frequency modes, which are sensitive to the conformat ion of the peripheral substituents on the heme, differ substantially f rom those in mammalian hemoglobins. The results show that in the dimer ic hemoglobin the interactions between the heme and the protein pocket are substantially different and are consistent with a mechanism of co operativity involving direct communication between the two hemes.