Antonio Tsuneshige

Electron Paramagnetic Resonance and Oxygen Binding Studies of α-Nitrosyl Hemoglobin A NOVEL OXYGEN CARRIER HAVING NO-ASSISTED ALLOSTERIC FUNCTIONS

α-Nitrosyl hemoglobin, α(Fe-NO)2β(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. α-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its β-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the α(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, α(Fe-NO)2β(Fe)2is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of α-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysβ93 toward 4,4′-dipyridyl disulfide of several α-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, α-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the α-subunits. It is concluded that the NO bound to the heme sites of the α-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions.

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery.

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features. These findings provide a new global view for allosteric interactions and invoke for a modern interpretation of the role of allosteric effectors and a reformulation of the Monod-Wyman-Changeaux model for control of allosteric systems, and other complementary models as well.

The global allostery model of hemoglobin: an allosteric mechanism involving homotropic and heterotropic interactions.

Studies of the allosteric mechanism of hemoglobin (Hb) have evolved from phenomenological descriptions to structure-based molecular mechanisms, as the molecular structures of Hb in deoxy and ligated states have been elucidated. The MWC two-state concerted model has been the widely accepted as the most plausible of the allosteric mechanisms of Hb. It assumes that the O2-affinity of Hb is regulated/controlled primarily by the T/R quaternary structural transition and that heterotropic effectors bind preferentially to T (deoxy) Hb to shift the T/R allosteric equilibrium toward the T state. However, recent more comprehensive O2-binding measurements of Hb have revealed a new mechanism, the Global Allostery model. It describes that the O2-affinity and the cooperativity are modulated in greater extents and the Bohr effect is generated primarily by the tertiary structural changes in both T (deoxy) and R (ligated) states of Hb. Differential interactions of heterotropic allosteric effectors with both T (deoxy) and R (ligated) states of Hb induce these tertiary structural changes. The X-ray structure of a complex of R (ligated) Hb with BZF, a potent heterotropic effector, has revealed the stereo-chemical influence of these effectors on the structure of R (ligated) Hb, resulting in the reduction of the ligand affinity in R (ligated) Hb. This model stresses the fundamental importance of the heterotropic interactions in regulation/control of the functionality of Hb. They alter the tertiary structures of both T (deoxy) and R (oxy) Hb, leading to large-scale modulations of the O2 affinity (KT and KR), and consequently the cooperativity (KR/KT) and the Bohr effect (delta P50/delta pH) from a global viewpoint of allostery in Hb.

Description of hemoglobin oxygenation under universal solution conditions by a global allostery model with a single adjustable parameter

The Monod-Wyman-Changeux allosteric model parameters evaluated from accurate oxygen equilibrium curves (OECs) of hemoglobin that were measured in an extremely wide range of structural constraints, imposed by allosteric effectors, yielded a closed circle when log K(T) and log K(R) were plotted against log L(0) and log L(4), respectively, showing novel phenomena that L(0) and L(4) have a maximal value and a minimal value, respectively, and K(T) and K(R) vary by more than three orders of magnitude. These phenomena were successfully described by a global allostery model, which mathematically keeps the frame work of the MWC model, but allows that K(T) under a set of solution conditions becomes larger than K(R) under another set of solution conditions and postulates that a representative allosteric effector binds to both the T and R states with a lower affinity but with a larger stoichiometry for the R state than for the T state. Thus, this global model can describe any given OEC measured under universal solution conditions with the single adjustable parameter, the concentration of the representative effector.

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl–, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (αFeO2)2-(βZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70–200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl–. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

OXYGENATION PROPERTIES OF HUMAN ERYTHROCYTES CONTAINING EXCLUSIVELY α-NITROSYL HEMOGLOBIN: A PROMISING BLOOD TRANSFUSANT CANDIDATE

We have prepared human erythrocytes that contain exclusively α-nitrosyl hemoglobin (Hb), i.e., α(Fe-NO)2β(Fe-O2)2, by incorporating nitric oxide (NO) into erythrocytes in a well-controlled nitrosylation process. The amount of α(Fe-NO) corresponding to 50% of the total heme content of the erythrocytes and exclusive binding of NO to α-subunits of intraerythrocytic Hb were confirmed by EPR. Oxygenation experiments on the intraerythrocytic α-nitrosyl Hb over a wide range of pH showed that: (1) the oxygen affinity of cell-free and intraerythrocytic α-nitrosyl Hbs were much lower than native Hb in their respective environments; (2) the oxygenation characteristics of the intraerythrocytic α-nitrosyl Hb in the acidic range was similar to that of the cell-free α-nitrosyl Hb in the presence of 2,3-diphosphoglycerate; and (3) the apparent Bohr effect in the intraerythrocytic α-nitrosyl Hb was dramatically diminished. This can be due to a restricted variation in intraerythrocytic pH in the alkaline region and the presence and/or production of endogenous 2,3-diphosphoglycerate. By comparing oxygen saturation characteristics, it was found that the intraerythrocytic α-nitrosyl Hb, despite its halved oxygen carrying capacity, could deliver more oxygen than DPG-depleted erythrocytes under similar experimental conditions. This makes α-nitrosyl Hb-containing erythrocytes a promising candidate for blood transfusant.

Preparation of mixed metal hybrids

Publisher Summary Mixed metal hybrid hemoglobin tetramers, α(Fe) 2 β(Mg) 2 , which carry natural protoheme (Fe) in the α subunits and other metallo(proto)porphyrins (Me) in the β subunits, and its complimentary form, α(Mg) 2 β(Fe) 2 , have been prepared and utilized in spectroscopic, functional, and structural studies of hemoglobin (Hb). These metalloporphyrins serve as functional and nonfunctional spectroscopic probes. These mixed metal hybrids allow to observe structural and functional properties of one type of subunit apart from their partner subunits, because properties of the α and β subunits carrying different types of metalloporphyrins are quite distinct. Mixed metal hybrids with combinations of Fe–Co, Fe–Ni, Fe–Zn, and Fe–Mn are typical examples that have been successfully studied. One principal method available for preparation of mixed metal hybrid Hbs is not only readily performed but is also widely applicable to many metalloporphyrin-containing Hbs. It consists of isolation of α and β subunits from two parent Hbs containing different prosthetic groups, followed by stoichiometric mixing of two appropriate subunits, which spontaneously form a mixed metal hybrid Hb tetramer.

A Novel Blood Transfusant Candidate: Intact Human Erythrocytes Containing Hemoglobin Exclusively Nitrosylated in the α-Subunits

The transport of oxygen from lungs to peripheral tissues is carried out by hemoglobin (Hb), the main protein component inside red blood cells. However, the modulation of its physiological performance is regulated by the intracellular content of metabolites, mainly 2,3-diphosphoglycerate (DPG), which lower the oxygen affinity of Hb to optimal levels, so that an efficient oxygen delivery is accomplished within a narrow gradient in partial pressures of oxygen between lungs and tissues. However, intracellular levels of DPG in stored blood decay with time, which causes a gradual increase in oxygen affinity, thus imposing a limit to the lifetime of blood for practical use.

The Intradimeric Alpha1beta1 Interface Holds the Key to Allosteric Control in Hemoglobin

The major quaternary structural change responsible for the allosteric transition in human adult hemoglobin (Hb) from low-affinity T- conformation to high-affinity R-conformation upon ligand binding to hemes has been described as a 15-degree rotation of one alpha1beta1 dimer over the pairing one. This interdimeric event has been in the limelight for decades. However, since very few changes, if any, have been reported for the alpha1beta1 intradimeric interface, its contribution to allostery has been neglected. Here, dramatic changes in allosteric characteristics that are caused by a specific steric alteration of the alpha1beta1 interface by chemical modification are reported.The chemically-modified alpha1beta1 interface Hb showed an extremely low affinity for oxygen and an almost complete absence of cooperativity, comparable to those caused in native Hb by a combination of strong allosteric effectors. The functional characterization is supported by structural evidence revealed by EPR using the nitrosyl derivative, which resembles that one characterized by an extremely low affinity for the ligand showing a triplet superhyperfine around g = 2. Sedimentation experiments showed that modified dimers associated tightly into tetramers. Calorimetric experiments, on the other hand, revealed that the stabilizing interactions were mainly hydrophobic.The above-mentioned findings suggest that the alpha1beta1 interface is not inert, but rather an extremely important interface playing a pivotal role on the control of oxygen affinity.

Contrasting Effects of Halides on the Structure and Function of A Multimeric Allosteric Protein

We have used two halide salts, namely, sodium chloride and sodium iodide, and studied their impact on the oxygenation characteristics of adult human hemoglobin (Hb). Previous studies in our group showed that both the halide salts exerted similar effects on the Hb function at concentrations below 0.1 M, i.e., an overall decrease in the affinity for oxygen, as a result of a decrease in the affinity at low oxygenation levels. However, as the halide concentrations increased, while chloride continued producing a progressive but rather diminished effect, iodide reverted its effects on Hb: the overall affinity for oxygen rather increased. Careful analysis of the oxygenation curves revealed that while the affinity for oxygen decreased at high oxygen saturation levels, the affinity at low oxygen concentration increased markedly. These effects reached a plateau at a concentration of 2 M, but even more surprisingly, cooperativity was never canceled. The results hinted at the possibility that iodide ions were splitting the tetrameric Hb molecules into asymmetric dimers. Dimers have been and still are considered non-cooperative, high oxygen-affinity systems. Yet, our present data clearly contrast with the previous tenet since cooperativity index showed values as high as 1.6 in the presence of 2 M NaI. Determination of molecular weight by size exclusion chromatography, and the study of oxygenation characteristics of symmetric nickel-iron Hb hybrids in the presence of sodium iodide showed that in fact the tetrameric Hb splits into two dimers that, strikingly, remain allosterically functional.