The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-270C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H20) and two exchangeable proton resonances (at alut -6.4 and -9.4 ppm from H20) as a function of oxygenation, the amount of oxygen bound to the a and /B chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential 02 binding to the a or chains; (ii) Despite considerable biochemical and biophysical studies devoted to the hemoglobin (Hb) molecule, the detailed molecular mechanism for the cooperative oxygenation of Hb is not fully understood. For example, it has not been possible, as yet, to correlate quantitatively various structural changes during the oxygenation process with the energetics of the process. A successful solution of the Hb problem is important not only because it would allow us to understand the molecular mechanism of a respiratory transport process, but also because it may give insights into the action of other more complex systems on which metabolic regulation depends. For recent reviews on the structure-function relationship in Hb, refer to refs. 1-5.Comparing the atomic models of deoxyhemoglobin and oxyhemoglobin-like methemoglobin, Perutz (6) has proposed a stereochemical mechanism for the cooperative oxygenation of Hb. In its original form, his model emphasized the link between the cooperativity and the transition between the two quaternary structures (deoxy quaternary structure is symbolized residues as excellent markers to monitor both tertiary and quaternary structural changes of Hb upon oxygenation. By choosing appropriate human mutant hemoglobins as well as chemically modified hemoglobins and by making use of Perutz's atomic models of hemoglobin, we have assigned a number of proton resonances, obtained through high-resolution proton nuclear magnetic resonance (NMR) spectroscopy, to specific amino acid residues in the Hb molecule. Of special interest to this work are the following resonances: the hyperfine shifted proton resonances at about -18 and -12 ppm from H20 due to protons from the # and a heme, respectively (8, 9); the exchangeable proton resonance at about -9.4 ppm from H20 due to the intersubunit hydrogen bond between a42(C7) tyrosine and f399(G1) aspartic acid in the a1/32 subunit interface, a characteristic feature of the deoxy quaternary structure (2, 10); and the exchangeable proton resonance at about -6.4 ppm from H20 due to the intrasubunit hydrogen bond between ,B98(FG5) valine and 3145(HC2) tyrosine, an important feature in the deoxy tertiary structure (11). Hence, b...

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.

Three members of an Oxfordshire family have polycythaemia. In each case their whole-blood oxygen affinity is increased. This is due to a previously undescribed haemoglobin variant which has been named haemoglobin Radcliffe (alpha2beta299(Gl)Ala). In addition to having a high oxygen affinity haemoglobin Radcliffe shows virtually no haem-haem interaction and a diminished Bohr effect. It is synthesized at the same rate and is as stable as haemoglobin A. X-ray analysis indicates that crystals of deoxyhaemoglobin Radcliffe are isomorphous with those of deoxyhaemoglobin A. Solutions of haemoglobin Radcliffe were also studied by high-resolution proton nuclear magnetic resonance spectroscopy. The structure/function relationships of haemoglobin Radcliffe are discussed in the light of these studies.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

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