title: Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions.
abstract: The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002248:Carbon Monoxide; D002263:Carboxyhemoglobin / Q000378:metabolism*; D002683:Chironomidae; D006418:Heme / Q000032:analysis; D006454:Hemoglobins / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D006454:Hemoglobins; D006418:Heme; D002248:Carbon Monoxide; D002263:Carboxyhemoglobin