New look at hemoglobin allostery.

Hemoglobin (Hb) is a truly remarkable molecule. Human adult hemoglobin (Hb A) has a tetrameric structure consisting of two α-chains with 141 amino acids each and two β-chains with 146 amino acids each. Figure 1 illustrates features of the molecule that will be discussed. The tertiary structure is the three dimensional structure of the individual protein chains. The quaternary structure is the arrangement of the multiple protein chains into a multi-subunit complex stabilized through non-covalent interactions. Each of the four chains in Hb possesses a heme group, the binding site for ligands, such as oxygen (O2), carbon monoxide (CO), or nitric oxide (NO). It is an essential protein for all vertebrates, designed to facilitate the loading of oxygen molecules in the lungs (or gills) and unloading of oxygen molecules in the tissues efficiently. Hb is one of the first proteins whose structure was determined by X-ray crystallography in the 1960s and has also been used as a paradigm for understanding the structure-function relationship in allosteric proteins. An allosteric protein is one in which binding of a substrate, product, or other effector to a subunit of a multi-subunit protein at a site (allosteric site) other than the functional site alters its conformation and functional properties and can therefore contribute to regulating its physiological properties. For a review of the structure-function relationship of Hb, see ref.1 Most of the published results and conclusions regarding the molecular basis for Hb function, until recently, were based on the information derived from X-ray crystallographic data of Hb, e.g., the classical Monod-Wyman-Changeux (MWC) and Perutz's two-structure stereochemical model for hemoglobin allostery.2-3 Figure 1 Structures of Hb A: (a) Crystal structure of deoxy-Hb A (2DN2); (b) Crystal structure of HbCO A (2DN3); (c) The 10 lowest-energy solution structures of HbCO A obtained by NMR spectroscopy (2M6Z); (d) Superimposition of the R (2DN3, colored in red), R2 ... The classical MWC/Perutz model postulates that all four subunits in Hb have to assume simultaneously either the tense (T)- or relaxed (R)-structure.2-3 Both structures can bind ligands while the affinity towards the ligand changes in transiting from the T- to the R-structure. Noticing the marked differences in the crystal structures of oxy- and deoxy-Hb, Perutz3 put forward his stereochemical mechanism that correlated the T- and R- states of the MWC model to the deoxy- and oxy-structures of Hb. A key feature of the MWC model is that all four subunits must make the switch from T to R or R to T at the same time. In other words, the ligation of one subunit would not affect the ligand affinity of the neighboring subunits within the same quaternary structure. It is a concerted quaternary structural transition model. Perutz's model further postulates that inter- and intra-subunit salt bridges stabilize the Hb molecule in the T-structure. The deoxy- or T-structure has a lower ligand affinity compared to the oxy- or R-structure and the binding of oxygen is cooperative, i.e., binding of the first oxygen molecule increases the affinity of the Hb molecule for additional oxygen molecules. The induced-fit or sequential model [also known as the Koshland-Nemethy-Filmer (KNF) model] is another classical model for Hb allostery.4-5 It postulates that the binding of a ligand to one subunit can induce the conformational changes in the tertiary structure of its neighboring subunits without their having a bound ligand. Thus, the ligand binding in a multi-subunit protein is a sequential process; there are not just two final states, T and R, but a series of intermediate states. A conformational change in a neighboring subunit can take place in the absence of ligand binding. Both the MWC and the KNF models can account for the cooperative oxygen binding to Hb, thus the ligand-binding data alone cannot distinguish the KNF model from the MWC/Perutz model. Much work has been done in the last sixty years in order to determine whether the transition from the T to the R state is concerted or sequential and to gain an understanding of the atomic and molecular details of the cooperative oxygenation of Hb A and the mechanism of allostery. The stereochemical mechanism of Perutz was extended by Szabo and Karplus6 and later refined by Lee and Karplus.7 This statistical-mechanical model derives a partition function that describes the influence of homotropic (oxygen) and heterotropic [e.g., hydrogen ions and 2,3-bisphosphoglycerate (2,3-BPG)] effectors on the Hb structural changes. Two different tertiary structures for each of the two quaternary structures have been included in their formulation. Contrary to Perutz's model, the Szabo-Karplus model takes into account the differences in strength of the salt bridges that stabilize the T-structure and the contributions of the pH-independent steric constraints in reducing the ligand affinity of Hb in the deoxy state. Yonetani and co-workers proposed a global allostery model.8 This model stipulates that in the absence of heterotropic effectors, the allostery of Hb follows the MWC/Perutz model. Heterotropic effectors, when present, interact with both T and R states of Hb to induce tertiary rather than quaternary structural changes. The changes in oxygen affinity, Bohr effect, and cooperativity of Hb are primarily the consequence of heterotropic effector-induced tertiary structural changes. The tertiary two-state model of Eaton and co-workers9 can be considered as a variation of the MWC/Perutz model. Within each quaternary structure, the subunits exist in equilibrium of high (r) and low (t) affinity conformers. The R- and T-structures as defined in the MWC/Perutz model favor the r and t formation, respectively. As in the MWC/Perutz model, ligand binding without a quaternary conformation change is non-cooperative. However, the tertiary conformations of individual subunits play the primary role instead of the quaternary conformations. The molecular code for cooperativity of Ackers and coworkers10-11 points out that there are eight ligation intermediates between the completely unliganded and the fully liganded tetrameric Hb. The tetrameric Hb switches from T- to R-form when at least one subunit of each dimer is liganded. Hence, five ligation intermediates plus the fully liganded tetrameric Hb exist in the R-structure. Within each quaternary state, oxygen binding or releasing “sequentially” modulates the tertiary constraints, which ultimately leads to the quaternary structural switch. Cooperativity is the result of both “concerted” quaternary switching and “sequential” modulation of ligand binding within each quaternary form. There are many crystal structures determined over the years and several well-characterized T- and R-types of crystal structures of Hb A reported in the literature are summarized in Table 1. This multitude of structures and recent results obtained by other methods clearly show that the classical two-structure MWC/Perutz description for hemoglobin allostery as presented in biochemistry, biophysics, and molecular biology textbooks cannot account for Hb function in details and needs revision. In our last review12 ten years ago, we gave a summary of our experimental results on the molecular basis of the Bohr effect of Hb A and the solution conformation, dynamics, and subunit communication of Hb A as derived from our nuclear magnetic resonance (NMR) studies. Here, we present new results of NMR and wide-angle X-ray scattering (WAXS) studies that are relevant to the structure-function relationship in hemoglobin. Table 1 Crystallization conditions and resolutions of various crystal structures. During the past 10 years, hemoglobin has remained an active research area for biochemical, biophysical, and computational studies with over 6,500 papers published in the literature. It is interesting to note that a search of PubMed indicates that there were 489 papers with hemoglobin titles published in 2004, 632 papers in 2009, and 813 papers in 2013. This increase in the number of Hb publications indicates that there are new results as well as new thinking in the field of hemoglobin research. This article is not intended to cover all areas of Hb research, but focuses on new findings on the nature of Hb as a dynamic ensemble as related to its properties in solution. For additional readings on Hb and Hb allostery, one could consider a number of relevant articles.13-31