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

Abstract

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the a 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 (3 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, a(Fe-CO)2J8(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 a 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.The simplest and conceptually most appealing theory that relates the structure of Hb to its cooperative behavior is the two-state allosteric model (1-5). According to this model, Hb exists in two affinity states-a low-affinity state having the quaternary structure of fully deoxygenated Hb, called T, and a high-affinity state with the quaternary structure of fully liganded Hb, called R. The essential feature of the model is that the affinity does not depend on the number of ligands bound per se, but is determined only by the quaternary structure (4). The two-state allosteric model was first applied to the equilibrium ligand-binding properties of Hb but naturally has been extended to explain cooperativity in the kinetics of both 02 and CO binding (6). Corresponding to the two affinities of the equilibrium treatment, there are four rates-one binding rate and one dissociation rate for each quaternary structure. For CO, the binding rate for the R state is about 60 times faster than for the T state (7), whereas the limited data suggest that the dissociation rate for the T state is about 10-fold greater than that for the R state (8). For 02, cooperativity is mainly manifested in the dissociation rates (9), with the T state dissociating about 150 times faster than the R state (10).Photodissociation experiments indicate that the mechanism is more complex than one in which binding of a ligand from the solvent or dissociation of a ligand from the heme into the solvent occurs in a single step (11,12). Both CO (13) and 02 (14,15) und...

New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels

“…When encapsulation was carried out in the deoxy form in the presence of the allosteric effectors inositol hexaphosphate and bezafibrate (T ϩ molecules) and followed by the addition of CO, the kinetics were again very similar to what was expected for hemoglobin in the T quaternary structure from studies on molecules free in solution (28). There is no geminate phase (Ͻ1%) and only a single bimolecular phase at Ϸ10 ms (Fig.…”

Probing the Hemoglobin Central Cavity by Direct Quantification of Effector Binding Using Fluorescence Lifetime Methods

“…Significantly, IHP decreases the yield of geminate recombination on the picosecond and nanosecond time scales (29,30), but not to the extent seen in the T state species (31). Upon addition of IHP, the visible resonance Raman spectra of the transient forms of HbA generated within 30 ps (32) and 10 ns (16,17) of photodissociating the parent HbACO show a decrease in the frequency of the iron-proximal histidine stretching mode from 230 cm Ϫ1 to ϳ226 cm Ϫ1 .…”

Crystal Structure of Horse Carbonmonoxyhemoglobin-Bezafibrate Complex at 1.55-Å Resolution

“…The basic assumption underlying the use of BZF for hemoglobin research has been that the drug interacts only to T-state hemoglobin (13)(14)(15)(16). However, some evidence shows that this assumption is not correct.…”