The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin

L. P. Murray, J. Hofrichter, E. R. Henry, M. Ikeda-Saito, K. Kitagishi, T. Yonetani, W. A. Eaton

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Abstract

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the α 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 β 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, α(Fe-CO)2β(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 <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 α 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.

Original languageEnglish
Pages (from-to)2151-2155
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number7
DOIs
Publication statusPublished - 1988

Keywords

  • allosteric control
  • cooperativity
  • laser photolysis
  • singular value decomposition
  • tertiary structure

ASJC Scopus subject areas

  • General

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