Observation of a stable intermediate form in the reaction of human hemoglobin with carbon monoxide

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e. 426 nm). As previously reported by others [Gray, R.D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176-5178], in the presence of 0.1 M phosphate pH 7.0 a rise-and-fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i.e. final [CO]/[heme] less than or equal to 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Y less than or equal to 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Y approximately 0.4 has a value similar to that observed starting from the fully liganded form. These results can be accounted for by a two-state allosteric model [Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the T0-R spectral changes but not for the T0-T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable.