Kinetic, structural, and spectroscopic indentification of geminate states of myoglobin: a ligand binding site on the reaction pathway

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Abstract
Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than .apprx. 100 K. Mb*CO, which is accumulated by photolysis between 1.2 and .apprx. 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., and Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and .apprx. 100 K, geminate states are also identified that have recombination rates of .apprx. 103 s-1 and .apprx. 10-5 s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb**, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb** is similar to that of Mb*CO. In both geminate states, the iron-proximal histidine distance remains unchanged (.+-. 0.02 .ANG.) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In Mb*CO the CO remains close to iron but not bound, and the Fe.cntdot..cntdot..cntdot.CO angle, which is bent in MbCO (127 .+-. 4.degree. C), is decreased by .apprx. 15.degree. [Powers, L., Sessler, J. L., Woolery, G. L., and Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb**, however, has moved approximately 0.7 .ANG. further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.