LIGAND-BINDING TO HEME-PROTEINS - THE EFFECT OF LIGHT ON LIGAND-BINDING IN MYOGLOBIN

Extended illumination slows the rebinding of CO tb myoglobin after pho todissociation at cryogenic temperatures. Two types of models have bee n put forward to explain the effect: motions of the CO within the heme pocket dr conformational transitions of the protein. To resolve this ambiguity, we have studied the effect of extended illumination on liga nd binding to horse and sperm whale myoglobin (hMb and swMb) with temp erature-derivative spectroscopy, monitoring the reaction in the CO str etch bands in the infrared and the conformation-sensitive band IU near 760 nm. The experiments show that the stretch frequency of the photod issociated CO does not change upon illumination, implying that the slo wing of the CO rebinding is caused by conformational relaxation of Mb from the bound state toward the deoxy structure. The light-induced rel axation (LIR) depends on the number of photons absorbed but not on the light intensity or duration separately. LIR occurs on photon absorpti on in either the bound or photodissociated state and depends on the te mperature at which the MbCO is illuminated. The LIR proceeds in jumps through a small number of conformational substates. The effective barr ier for rebinding increases with each step. The substates populated ar e similar to those found in the thermally-induced relaxation (TIR) tha t is observed above 160 K. LIR depends markedly on the structural deta ils; it differs for swMbCO and hMbCO and even for the three A substate s of swMbCO. Pronounced differences exist between the effects in MbCO and MbO(2). The similarity of LIR and TIR leads to a revised model for ligand binding to swMbCO and hMbCO, in which the relaxation is crucia l for the escape of the ligand from the pocket, as was first suggested by Friedman [Friedman, J. M. (1985) Science 228, 1273-1280].