Mechanism of the control of dioxygen binding in a dimeric cobalt-substituted insect hemoglobin. Resonance Raman evidence for cobalt-axial-ligand bond changes.
Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt-substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH-induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand-cobalt bonds. Using 16O2/18O2 isotope substitution the O-O and Co-O2 stretching and the Co-O-O bending mode have been assigned to the two affinity states of this hemoglobin: v (O-O) changes from 1152 cm-1 (pH 5.5; t conformation) to about 1125 cm-1 (pH 9.5, r conformation), v (Co-O2) from 512 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) and delta (Co-O-O) from 378 cm-1 (pH 5.5) to 390 cm-1 (pH 9.5). The Co-N epsilon (His) stretching mode has also been detected changing from 313 cm-1 (pH 5.5) to 307 cm-1 (pH 9.5). For the first time, reciprocal behaviour between the Co-N epsilon and Co-O2 bonds and between the Co-O2 and the O-O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411-415 cm-1 has been investigated and shown also to reflect the t in equilibrium with r conformation transition.
D000818:Animals; D002683:Chironomidae / Q000032:analysis*; D003035:Cobalt / Q000378:metabolism*; D004175:Diptera / Q000032:analysis*; D006454:Hemoglobins / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
D006454:Hemoglobins; D046911:Macromolecular Substances; D003035:Cobalt; D006639:Histidine; D010100:Oxygen