Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway.
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 approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 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 MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A 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.