Dynamics of the quaternary conformational change in trout hemoglobin.
The kinetics of conformational changes in trout hemoglobin I have been characterized over the temperature range 2-65 degrees C from time-resolved absorption spectra measured following photodissociation of the carbon monoxide complex. Changes in the spectra of the deoxyheme photoproduct were used to monitor changes in the protein conformation. Although the deoxyheme spectral changes are only about 8% of the total spectral change due to ligand rebinding, a combination of high-precision measurements and singular value decomposition of the data permits a detailed analysis of both their amplitudes and relaxation rates. Systematic variation of the degree of photolysis was used to alter the distribution of liganded tetramers, permitting the assignment of the spectral relaxation at 20 microseconds to the R----T quaternary conformational change of the zero-liganded and singly liganded molecules and spectral relaxations at about 50 ns and 2 microseconds to tertiary conformational changes within the R structure. Analysis of the effect of photoselection by the linearly polarized excitation pulse indicates that a major contribution to the apparent geminate rebinding in the 50-ns relaxation arises from rotational diffusion of molecules containing unphotolyzed heme-CO complexes. The activation enthalpy and activation entropy for the R0----T0 transition are +7.4 kcal/mol and -12 cal mol-1 K-1. Using the equilibrium data, delta H = +29.4 kcal/mol and delta S = +84.4 cal mol-1 K-1 [Barisas, B. G., & Gill, S. J. (1979) Biophys. Chem. 9, 235-244], the activation parameters for the T0----R0 transition are calculated to be delta H = +37 kcal/mol and delta S = +73 cal mol-1 K-1. The similarity of the equilibrium and activation parameters for the T0----R0 transition indicates that the transition state is much more R-like than T-like. This result suggests that in the path from T0 to R0 the subunits have already almost completely rearranged into the R configuration when the transition state is reached, while in the path from R0 to T0 the subunits remain in a configuration close to R in the transition state. The finding of an R-like transition state explains why the binding of ligands causes much smaller changes in the R----T rates than in the T----R rates.
Hofrichter
J
J
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Henry
E R
ER
Szabo
A
A
Murray
L P
LP
Ansari
A
A
Jones
C M
CM
Coletta
M
M
Falcioni
G
G
Brunori
M
M
Eaton
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D002263:Carboxyhemoglobin / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013816:Thermodynamics; D014337:Trout
D006454:Hemoglobins; D046911:Macromolecular Substances; D002263:Carboxyhemoglobin