Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.

Abstract

The photodissociation of CO from HbCO at ambient temperature is studied by means of a femtosecond IR technique. The bleaching of the FeCO absorption and the appearance of a new IR absorption near that of free CO are both observed at 300 fs after optical excitation. The bleach does not recover on the time scale of a few picoseconds but does recover by ;7--4% within 1 ns, which suggests that a barrier to recombination is formed within a few picoseconds. The CO spectrum does not change significantly between 300 fs and 1 ns, suggesting that the CO quickly finds some locations in the heme pocket that are not more than a few angstroms from the iron. The de-ligated CO appears in its ground vibrational level. There is evidence that 85 ± 10% of this CO remains in the heme pocket at 1 ns; it probably resides there for 50 ns. The flow of excess vibrational energy from the heme to the solvent was directly observed in the IR experiments. The heme cools within 1-2 ps while thermal disruption of the surrounding solvent structure requires ""30 ps.Ultrafast spectroscopic methods have contributed much to understanding the photophysics and structural dynamics that occur after the photodissociation of oxy-and carboxyhemoglobin (1-3). The motion of the iron out of the heme plane has been discussed in some detail already, and evidence from optical spectroscopy (3) and Raman scattering (4, 5) suggests that the heme is partially or fully domed within 350 fs and perhaps as rapidly as 50 fs. This time frame for motion of the iron and its associated proximal histidine is also found in theoretical simulations of the heme relaxation (6). On the other hand, very little is yet known about the properties of the ligand generated by photolysis in ambient temperature solution.In the normal operation of hemoglobin, 02 diffuses into the protein from the solvent and bonds to the iron. Questions regarding ligand diffusion into and escape from the heme pocket as well as binding within the protein therefore have central importance to the function of hemoglobin. The hemoglobin structure, as determined from x-ray analysis (7), has no channel large enough to facilitate passage of a ligand between the solvent and heme. Hence, diffusion to the heme requires significant globin motions (7-9). Although transient optical methods are sensitive, the electronic spectrum of hemes cannot respond to changes in the location of a relatively inert diatomic molecule within the protein. Thus, experiments that can observe the ligand directly and monitor any changes in its structure or environment are expected to extend significantly our understanding of hemoprotein function. Such measurements on free CO would enable us to address some new issues for hemoglobin under ambient aqueous conditions. For example, additional information could be obtained on the rate of generation of CO and on the pathways of photodissociation (10, 11), the efficiency of subnanosecond geminate recombination of CO to iron could be brought into relation with results from nanosecond (12) and m...

Ultrafast anisotropic protein quake propagation after CO photodissociation in myoglobin

“…The dissociation kinetics was similar to previous studies, characterized by cooling of the CO within 300 fs due to collisions with protein atoms, and a cooling of the heme within 1-2 ps, followed by slower relaxations (Fig. S1) (5,10).…”

“…Because the CO-iron bonds in a Mb ensemble can be coherently photolysed with a laser flash within only 50 fs (4), a range of time-resolved techniques has been applied to probe various aspects of the structural dynamics after CO photodissociation, such as energy dissipation, CO dynamics, or heme doming (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Three-dimensional movies of Mb conformational transitions were derived by means of time-resolved crystallography, which recently reached subpicosecond time resolution (16)(17)(18)(19).…”

Multiply Excited Vibration of Carbon Monoxide in the Primary Docking Site of Hemoglobin Following Photolysis from the Heme

“…The heme proteins studied in most detail are myoglobin and hemoglobin, pioneered in ultrafast MIR studies by Anfinrud, Lim, and co-workers for the ligand CO. They observed the bleaching of carboxyhemoglobin (HbCO) after photolysis 1 and the CO absorption in the primary docking site of carboxymyoglobin (MbCO) and HbCO, 2 where the CO exists in two (presumably antiparallel) orientations parallel to the heme plane and hence almost perpendicular to the Febound orientation. [3][4][5] Furthermore,arisetimeforthedockingsite absorption signal 6 and a spectral evolution due to interconversion of the two CO orientations 7 and to conformational changes of the protein 8 were reported, while the total amount of docked CO remained constant for hundreds of picoseconds.…”

Docking Site Dynamics of ba 3-Cytochrome c Oxidase from Thermus thermophilus

“…Both the B 0 and B 1 states in cytochrome ba 3 have ⌬1 ⁄2 ϳ12 cm Ϫ1 indicating that the B 0 state arises from CO that remains in the docking site. If the CO ligand corresponding to the B 0 state had been laid outside the docking site as solvated, its detection would have been difficult because the spectrum of CO in H 2 O is too diffuse to be observed (5,7). Despite the close resemblance in the energies of the B 1 and B 0 states of unligated CO, between Mb and cytochrome ba 3 there are profound differences in the dynamic properties of their docking sites.…”

“…Time-resolved x-ray and time-resolved infrared experiments of photolyzed CO-bound myoglobin (Mb) 1 have demonstrated the existence of docking sites and their relevance in physiological ligand binding (1)(2)(3)(4)(5)(6)(7)(8). However, the different trajectories of the funneled photodissociated ligand in the docking site can be distinguished only through time-resolved infrared spectroscopy (2)(3)(4)(5). There is now a relatively good understanding of the ligand dynamics of photolyzed Mb-CO. To date Mb has played the role of a model system for determining docking sites and channels through which ligands enter and escape from the active site of proteins, establishing a foundation for their chemical dynamics (9 -11).…”