Evolution of allosteric models for hemoglobin

Abstract

SummaryWe compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)-and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond -millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.IUBMB Life, 59: 586-599, 2007

Experimental basis for a new allosteric model for multisubunit proteins

“…Our finding of two functional conformations for the subunits of hemoglobin in each quaternary structure is readily explained by the simplest possible extension of the allosteric model of MWC to include tertiary equilibria, and is inconsistent with all other theoretical models proposed for hemoglobin (12). In this TTS allosteric model (Fig.…”

“…The second postulate is that allosteric effectors regulate oxygen affinity by altering only the R ⇄ T preequilibrium. Investigations of hemoglobin focused primarily on the first postulate, which was finally confirmed by single-crystal oxygen-binding measurements that ruled out a sequential model (8) and ended a 25-y controversy (9)(10)(11)(12). The second is of more general interest because it applies to all multisubunit proteins exhibiting allosteric behavior and has been known for many years to be inconsistent with the fact that allosteric effectors markedly affect oxygen affinity without changing the quaternary preequilibrium [see data summaries by Imai, Yonetani, and coworkers (13,14)].…”

“…The TTS model has also been used by Spiro and coworkers (39) to explain the kinetics of structural changes observed by time-resolved resonance Raman experiments. To distinguish between the TTS model and other theoretical models for hemoglobin allostery (12), it became essential to determine the functional properties of the t subunits of unliganded R. We therefore designed a new kind of laser photolysis experiment that consists of extended cw illumination of HbCO encapsulated in the R quaternary structure to create an equilibrium population of unliganded subunits. It was expected that the gel would also prevent tertiary conformational exchange during ligand rebinding, so that the rebinding kinetics of individual conformations could be measured in the absence of any tertiary conformational exchange dynamics.…”

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin

“…The much larger structural changes of the quaternary allosteric transition (involving shifts in the inter-subunit contacts and relative motions of the two symmetric dimers α 1 β 1 ∕α 2 β 2 ) occur in the microsecond time range; the single time constant estimated from optical changes of the deoxy photoproduct is about 20 μs for human HbA at neutral pH and 20°C (2,3,6,7). The dynamics of the structural changes followed by ultraviolet resonance Raman (UVRR) spectroscopy after laser photolysis showed, however, the quaternary conformational transition involving contacts at the α 1 β 2 and α 2 β 1 interfaces (8, 9) to be more complex.…”

“…Changes in the optical and resonance Raman spectra of the different states have provided, over the last four decades, a quantitative estimate of the rates of the competing events (2)(3)(4)(5). For a review on time-resolved optical absorption (TR-OA) data describing conformational decays as well as rebinding in the dark of a ligand escaped into the solvent (bimolecular) or trapped within the protein matrix (geminate), see Eaton et al (6).…”