Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: direct evidence for the functional significance of a hierarchy of dynamical processes.
Band III is a disorder and conformation-sensitive near-infrared (approximately 760 nm) charge transfer absorption band characteristic of equilibrium and nonequilibrium five coordinate ferrous high-spin hemes. The time evolution of this absorption band subsequent to photodissociation of six coordinate ferrous hemoglobin or myoglobin can provide detailed information regarding conformational relaxation, including the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic. Such time-resolved measurements over a range of temperatures are difficult due to long sample recovery times at cryogenic temperatures. A new restoring technique that allows for the rapid movement of a large optically accessible cryostat is used in combination with nanosecond time-resolved near-infrared absorption spectroscopy to generate band III as a function of time for the photoproducts of the carbon monoxide derivative of adult human hemoglobin (COHbA) and, to a more limited extent, horse myoglobin (COMb). The measurements are made over a wide range of temperatures extending from well below the solvent (75% glycerol:water) glass transition at approximately 180 K to ambient temperatures. Three temperature- and/or viscosity-dependent phenomena are observed. At the highest temperatures, only conformational relaxation is observed for the 75% glycerol sample. At very high viscosity (> or = 400 cp), conformational relaxation slows dramatically, and both kinetic hole burning followed by the filling in of the "hole" (dynamic hole filling) are observed. As the temperature is lowered, conformational relaxation slows and finally ceases. Kinetic hole burning and dynamic hole filling as well as additional broadening of band III are observed down to 140 K. The observation of kinetic hole burning (KHB) is indicative of the sample being inhomogeneous on the time scale of the ligand rebinding giving rise to KHB. The onset of hole filling is a direct manifestation of the thermal homogenization of the initial inhomogeneous distribution of conformational substates responsible for KHB. The observed dynamics are used to explain the inverse temperature effect associated with the non-Arrhenius slow down of geminate rebinding above approximately 180 K. The inverse temperature effect appears to arise not only from the onset of conformational relaxation but also from the increase in the rate on thermal averaging of the initial inhomogeneous distribution of conformational substates.
Huang
J
J
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Ridsdale
A
A
Wang
J
J
Friedman
J M
JM
D000328:Adult; D000818:Animals; D002263:Carboxyhemoglobin / Q000737:chemistry / Q000378:metabolism; D006418:Heme; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D006441:Hemoglobin A / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D006801:Humans; D007700:Kinetics; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics; D013997:Time Factors
D006420:Hemeproteins; D009211:Myoglobin; C018013:carboxymyoglobin; D006418:Heme; D006441:Hemoglobin A; D002263:Carboxyhemoglobin