title: Investigation of laser-induced long-lived states of photolyzed MbCO.
abstract: We present evidence from resonance Raman and absorption measurements that the extended exposure of MbCO to CW laser light at low temperatures alters the CO rebinding kinetics and leads to a significantly increased population of very long lived states of photolyzed MbCO. This optical "pumping" process is observed for samples frozen in both aqueous buffer and glycerol/buffer and exhibits power law behavior with a very weak temperature dependence. A comparison of the nonexponential rebinding kinetics of CO molecules from the pumped states with the rebinding observed in flash photolysis experiments suggests that the pumped states are distinct geminate states, not observed in flash photolysis experiments. Thus, a four-state model, with two geminate states, is implicated for MbCO. Pumped states may represent "separated geminate pair" states with the CO molecule still in the heme pocket or possibly trapped within a cavity on its way through the protein matrix, consistent with molecular dynamics simulations. The possibility of significant deoxyheme relaxation from a less domed to a more domed configuration, as a result of the multiple photolysis events associated with the pumping process, is also explored. However, the small changes observed in the Soret band line shape and position subsequent to pumping at T less than 180 K tend to rule out this explanation for the pumping process. Since the yield for creating a pumped state is small (e.g., less than 10(-7) for T greater than 100 K), pumping can be observed only after extended illumination and is absent in flash photolysis measurements, even after multiple flashes. At higher temperatures (T greater than 180 K), the escape of the CO molecule to the solvent is observed. Our data are consistent with a "phase transition" of the protein that is coupled to the surrounding matrix. The protein fluctuations are quenched below approximately 185 K for a solvent composed of 70% glycerol and below approximately 260 K for aqueous buffer. We also present the first large amplitude measurements of CO rebinding from the protein exterior, observed below 200 K after freezing the sample under laser illumination.
authors.affiliation: Department of Physics, Northeastern University, Boston, Massachusetts 02115.
authors.forename: V
authors.initials: V
authors.lastname: Srajer
authors.forename: L
authors.initials: L
authors.lastname: Reinisch
authors.forename: P M
authors.initials: PM
authors.lastname: Champion
mesh_terms: D000818:Animals; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008433:Mathematics; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D010782:Photolysis; D011485:Protein Binding; D012997:Solvents; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D013997:Time Factors; D014907:Whales
chemical_list: D009211:Myoglobin; D012997:Solvents; C018013:carboxymyoglobin; D006418:Heme