Initial trajectory of carbon monoxide after photodissociation from myoglobin at cryogenic temperatures.

Migration of the CO ligand following photolysis of carbonmonoxy myoglobin (MbCO) in single crystals has been investigated by time-resolved X-ray diffraction at 40K. After short illumination by weak visible light at a photolysis rate of approximately 1 s-1, the photodissociated CO molecule is found about 1 A from its bound location. After continuous illumination over several hours, the CO molecule migrates to a more distant site in the distal pocket, about 2.5 A from its bound location. Migration of the ligand under continuous illumination accounts for different locations of the photodissociated CO molecule previously reported in three cryocrystallographic studies [Teng, T.-Y., et al. (1994) Nat. Struct. Biol. 1, 701-705; Schlichting, I., et al. (1994) Nature 371, 808-812; Hartmann, H., et al. (1996) Proc.Natl.Acad. Sci. U.S.A. 93, 7013-7016]. Due to the different photolysis protocols employed in these studies, each reveals a different part of the trajectory of the photodissociated CO molecule. When the different experimental parts of the trajectory at 40 K are pieced together and combined with our nanosecond time-resolved studies at room temperature [Srajer, V., et al. (1996) Science 274, 1726-1729], excellent agreement is obtained with recent theoretical predictions of the CO probability distribution in the ligand pocket [Vitkup, D., et al. (1997) Nat. Struct. Biol. 4, 202-208]. The heme relaxation that accompanies ligand photolysis is incomplete, about 30% of that associated with the conversion of MbCO to deoxy-Mb at room temperature, and independent of the duration of illumination. Other tertiary structural changes in the globin are also greatly diminished. The globin structure is therefore very rigid at cryogenic temperatures, and structural relaxation is greatly hindered, consistent with numerous spectroscopic measurements.