Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy

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The haem-rotational disorder (insertion of haem into globin rotated about the α, γ-meso axis by 180 °) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two FeCN bending vibrations at 401 cm-1 and 422cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One FeGN bending mode at 422cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t → r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the FeNε(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single FeCN bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the FeCN bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the FeCN and vinyl bending modes. The Fe-Nε(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe 57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange. © 1987.

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Journal of Molecular Biology

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