柴山 修哉

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O-2] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O-2)beta(Fe-O-2)][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O-2 binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O-2 equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O-2-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2) were measured by using an Imai apparatus. It was shown that the first and second O-2 equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg-1, respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O-2 binding study.

The main features of cooperative oxygenation human hemoglobin have been described by assuming the equilibrium between two affinity conformations of the entire molecule, T and R, However, the molecular basis for explaining the wide variation in the O-2 affinities of the deoxy T state has remained obscure, We address this long-standing issue by trapping the conformational states of deoxyhemoglobin molecules within wet porous transparent silicate sol-gels. The equilibrium O-2 binding measurements of the encapsulated deoxyhemoglobin samples showed that deoxyhemoglobin free of anions coexists in two conformations that differ in O-2 affinity by 40 times or more, and addition of inositol hexaphosphate to this anion-free deoxyhemoglobin brings about a very slow redistribution of these affinity conformations, These results are the first, direct demonstration of the existence of equilibrium between two (at least two) functionally distinguishable conformational states in the T state deoxyhemoglobin. (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha(2)(Fe)beta(2)(M) and alpha(2)(M)beta(2)(Fe); M = Mg(II),Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha(2)(Fe)beta(2)(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effecters, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha(2)(Fe)beta(2)(Mg) and alpha(2)(Fe-CO)beta(2)(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K-1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effecters is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution. We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins. (C) 1999 Academic Press.

A controversy still exists over whether the molecular basis of hemoglobin cooperativity can be more appropriately explained by one of two classic allosteric models, the concerted and sequential models. To distinguish these two models from the viewpoint of their fundamental processes, namely, the presence or absence of conformational equilibria, we have trapped the conformations of nickel(II)-iron(II) hybrid hemoglobin molecules with two CO-bound, α2(Fe-CO)β2(Ni) and α2(Ni)β2(Fe-CO), by encapsulation in the water-filled pores of sol-gel-derived transparent silica-gels. In our experimental system, nickel(II) protoporphyrin binds neither O2 nor CO and mimics a fixed deoxyheme, and the gel matrix provides a means of inhibiting large-scale protein structural changes, thus enabling O2 equilibrium study of the hybrids still in their doubly liganded conformations. Results showed that two conformations of widely different O2 affinity exist together in each doubly liganded hemoglobin, providing a direct proof of the concerted mechanism versus the sequential mechanism.

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual alpha and beta subunits in human hemoglobin. To address this question systematically, we prepared a series of hybrid hemoglobins in which heme was replaced by chromium(III), manganese(III), nickel(II), and magnesium(II) protoporphyrin IXs in either the alpha or beta subunits to produce alpha(2)(M)beta(2)(Fe)(1) and alpha(2)(Fe)beta(2)(M) tetramers. None of the abnormal metal complexes react with dioxygen or carbon monoxide. The O-2 affinities of the resultant hemoglobins vary from 3 mu M-1 (Cr(III)/Fe(II) hybrids) to 0.003 mu M-1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A(0). The alpha and beta subunits in hemoglobin have similar O-2 affinities in both quaternary states, despite the fact that the R to T transition causes significantly different structural changes in the alpha and beta heme pockets, This functional equivalence almost certainly evolved to maintain high n values for efficient O-2 transport.

Cr(III)-Fe(II) hybrid hemoglobins, alpha(2)(Cr)beta(2)(Fe) and alpha(2)(Fe)beta(2)(Cr), in which hemes in either the alpha- or beta-subunits were substituted with chromium(III) protoporphyrin IX (Cr(III)PPIX), were prepared and characterized by oxygen equilibrium measurements. Because Cr(III)PPM binds neither oxygen molecules nor carbon monoxide, the oxygen equilibrium properties of Fe(II) subunits within these hybrids can be analyzed by a two-step oxygen equilibrium scheme. The oxygen equilibrium constants for both hybrids at the second oxygenation step agree with those for human adult hemoglobin at the last oxygenation step (at pH 6.5-8.4 with and without inositol hexaphosphate at 25 degrees C). The similarity between the effects of the Cr(III)PPM and each subunits' oxyheme on the oxygen equilibrium properties of the counterpart Fe(II) subunits within hemoglobin indicate the utility of Cr(III)PPM as a model for a permanently oxygenated heme within the hemoglobin molecule. We found that Cr(III)-Fe(II) hybrid hemoglobins have several advantages over cyanomet valency hybrid hemoglobins, which have been frequently used as a model system for partially oxygenated hemoglobins. In contrast to cyanomet heme, Cr(III)PPM within hemoglobin is not subject to reduction with dithionite or enzymatic reduction systems. Therefore, we could obtain more accurate and reasonable oxygen equilibrium curves of Cr(III)-Fe(II) hybrids in the presence of an enzymatic reduction system, and we could obtain single crystals of deoxy-alpha(2)(Cr)beta(2)(Fe) when grown in low salt solution in the presence of polyethylene glycol 1000 and 50 mM dithionite.

The geminate and bimolecular binding of CO, O-2, and NO to [alpha-Ni(II)](2)-[beta-Fe(II)](2) and [alpha-Fe(II)](2)-[beta-Ni(II)](2) hybrid hemoglobins has been studied. Bimolecular reactions: At pH 6.6 and 20 degrees both hybrids bind CO at 0.15 x 10(6) M(-1) s(-1). Reactions with oxygen: At pH 6.6 the on rates are 4.8 and 7.5 x 10(6) M(-1) s(-1) for alpha- and beta-hybrids, respectively; the off rate is approximately 2 x 10(3) s(-1) for both. At pH 8 the alpha-Fe hybrid shows cooperativity whereas the beta-hybrid does not. Nanosecond geminate reactions: Faster bimolecular rates correlate with larger, geminate amplitudes; thus alpha-Fe hybrids have larger amplitudes, and O-2 geminate amplitudes are larger than these with CO. At pH 8, 50% of O-2 recombines with the alpha-hybrid. With NO, nanosecond geminate recombination is observable only with the beta-hybrid. Picosecond reactions: alpha-Hybrids show picosecond recombination of O-2. With NO, alpha-hybrids recombine at 30 ns(-1), beta-hybrids at 0.3 ns(-1). The NO picosecond rates correlate with the molecular dynamics which shows ligands leaving the beta-Fe atom early and regularly, but remaining near the alpha-Fe atom. The results may be explained by assuming an interaction between the alpha-subunits giving rise to a high-affinity fast-reacting form, whereas the beta-subunits only become fast-reacting when an R-T conformation change analogous to that of hemoglobin A takes place. A third allosteric state is postulated to explain the results.

Copper(II)-iron(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunits are substituted with copper(II protoporphyrin IX, have been prepared, The affinities of the ferrous-subunits in both hybrids for the first binding oxygen are as low as the affinity of deoxyhemoglobin under various solution conditions, indicating the equality of behavior in copper(II) protoporphyrin IX and deoxyheme, Electron paramagnetic resonance (EPR) examinations on these hybrids at room temperature show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the alpha subunits within a low affinity conformation of hemoglobin, These results suggest that copper(II) protoporphyrin IX is a useful EPR probe at room temperature for investigating the deoxyheme environment in hemoglobin.

We have used the sol-gel method to encapsulate oxy- and deoxy haemoglobins in transparent wet porous silica gels and fixed their original functional states with the retention of the reversible oxygenation properties as well as the intact spectroscopic properties. Haemoglobin originally encapsulated in aerobic gel binds oxygen non-cooperatively with very high affinity, corresponding to that for the last oxygen molecule binding to haemoglobin in solution. In contrast, haemoglobin originally encapsulated in anaerobic gel binds oxygen non-cooperatively with very low affinity, comparable to that for the first oxygen molecule binding to haemoglobin in solution. Furthermore, a detailed comparison of visible absorption spectra of deoxygenated haemoglobins originally encapsulated in aerobic and anaerobic gels indicates the retention of their original quaternary structures during the oxygenation or deoxygenation process. These results demonstrate that oxygen affinities of oxy- and deoxyhaemoglobins in solution can be satisfactorily fixed by encapsulation in wet porous silica gels, which presumably prevents the changes in the quaternary structures of haemoglobin. In addition, these results suggest a new capability of the sol-gel method to control the structural states of a variety of proteins, and further open up a new area of investigation of protein structure-function relationships. (C) 1995 Academic Press Limited

The reaction of cyanide metmyoglobin (Mb+CN-) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb+CN- and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb+CN- and the intermediate are very similar, including the intensity ratios of the peak C1 to D. This indicates that CN- remains bound with a linear FeCN configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb+CN-, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb+CN- but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 ± 0.01 A ̊ for both Mb+CN- and the intermediate and 2.05 ± 0.01 A ̊ for deoxyMb. These results imply that the local structure around the heme iron of Mb+CN- does not change upon reduction until the cyanide ligand is released. © 1993.

The reaction of cyanide metmyoglobin (Mb+CN-) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb+CN- and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb+CN- and the intermediate are very similar, including the intensity ratios of the peak C1 to D. This indicates that CN- remains bound with a linear Fe-C-N configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb+CN-, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb+CN- but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 +/- 0.01 angstrom for both Mb+CN- and the intermediate and 2.05 +/- 0.01 angstrom for deoxyMb. These results imply that the local structure around the heme iron of Mb+CN- does not change upon reduction until the cyanide ligand is released.

Asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha(Fe)beta(Fe)][alpha(Ni)beta(Ni)], in which the alpha1beta1 dimer containing ferrous protoporphyrin IX and the complementary alpha2beta2 dimer containing Ni(II) protoporphyrin IX were cross-linked between Lys-82beta1 and Lys-82beta2 by reaction with bis(3,5-dibromosalicyl) fumarate, was synthesized and characterized. We have previously shown that (i) Ni(II) protoporphyrin IX, which binds neither oxygen nor carbon monoxide, mimics a fixed deoxyheme with respect to its effect on the oxygen equilibrium properties of the counterpart iron subunits in both symmetric Ni(II)-Fe(II) hybrid Hbs [Shibayama, N., Morimoto, H., & Miyazaki, G. (1986) J. Mol. Biol. 192, 323-329] and (ii) the cross-linking used in this study little affects the oxygen equilibrium properties of hemoglobin [Shibayama, N., Imai, K., Hirata, H., Hiraiwa, H., Morimoto, H., & Saigo, S. (1991) Biochemistry 30,8158-8165]. These remarkable features of our model allowed us to measure the oxygen equilibrium curves for the first two steps of oxygen binding to the alpha1beta1 dimer within the hemoglobin tetramer. At all pH values examined, the affinities of this asymmetric hybrid for the first oxygen molecule are as low as those of native hemoglobin. The hybrid did not show cooperative oxygen binding at pH 6.4, while significant cooperativity was observed with rising pH; i.e., the Hill coefficient was increased from 1.41 to 1.53 upon a pH change from 7.4 to 8.4. The electronic absorption spectrum of Ni(II) protoporphyrin IX in the alpha2 subunit was changed upon carbon monoxide (or oxygen) binding to the alpha1beta1 dimer. This change provides additional information about the interaction between liganded and unliganded dimers within the asymmetric tetramer.

The reaction of cyanomet myoglobin (Mb+CN-) with dithionite follows biphasic kinetics with formation of an unstable intermediate, supposed to be cyanide-bound ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using newly-developed rapid-freezing apparatus and compared with the spectra of Mb+CN- and deoxy myoglobin (Mb). Except for changes in the preedge peaks and a shift of the edge position, no significant changes are observed between the XANES spectra of Mb+CN- and the intermediate. This indicates that CN- remains bound linearly in the intermediate similarly as in Mb+CN-. The EXAFS of the intermediate is almost identical to that of Mb+CN- but different from that of deoxy Mb. Analysis of the EXAFS has shown that the average iron-nearest neighbor atom distance is 1.99 +/- 0.01 angstrom for both Mb+CN- and the intermediate and 2.05 +/- 0.01 angstrom for deoxy Mb. These results show that the local structure around the heme iron does not change upon reduction until the cyanide ligand is released.

Polymerization of half-liganded Hb S was investigated using Ni(II)-Fe(II) hybrid Hb S, in which heme in either alpha or beta-s subunits is replaced by Ni (II) protoporphyrin IX. Studies on the polymerization of these hybrid hemoglobins were carried out under aerobic conditions. Both alpha-2(Ni)beta-2-s(Fe-CO) and alpha-2(Fe-CO)beta-2-s(Ni) polymerized with a distinct delay time as do native deoxy-Hb S and Ni(II) Hb S. However, the critical concentration for polymerization of half-liganded Hb S, alpha-2(Ni)beta-2-s(Fe-CO) and alpha-2(Fe-CO)beta-2-s (Ni), was 4- and 8-times higher, respectively, than that of Ni(II)-Hb S. Kinetics of polymerization of both deoxygenated hybrid hemoglobins with CO completely removed were the same, although the critical concentrations for polymerization were intermediate between those for deoxy-Hb S and Ni(II)-Hb S. These results suggest that the small tertiary conformational change associated with the doubly liganded state may be much less favorable to polymerization than the completely unliganded state of Hb S. The conformational change depends on whether alpha or beta chain is liganded. The ease of polymerization and low solubility of sickle hemoglobin is dependent not only on quaternary, but on tertiary structural changes, as well as on the substitution of Val for Glu at the beta-6 position.