The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.
To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.
Murray
L P
LP
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Hofrichter
J
J
Henry
E R
ER
Ikeda-Saito
M
M
Kitagishi
K
K
Yonetani
T
T
Eaton
W A
WA
D000494:Allosteric Regulation; D002248:Carbon Monoxide / Q000378:metabolism*; D002263:Carboxyhemoglobin / Q000378:metabolism*; D003035:Cobalt; D006454:Hemoglobins / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature
D006454:Hemoglobins; D003035:Cobalt; D002248:Carbon Monoxide; D002263:Carboxyhemoglobin