柴山 修哉

X-ray fluorescence holography (XFH) is a technique that can directly image the 3D arrangement of atoms around an element in a sample. The holograms contain both intensity and phase information, allowing atomic reconstruction without needing prior structural information or a tentative structural model. XFH has already been used to reveal the local structures of various inorganic samples, and recently, work has begun on XFH for soft matter. In this paper, we review the progress of XFH on soft materials. First, we review the fundamental principles of XFH. Second, we review inverse mode XFH on soft materials, and the results of the experiments on hemoglobin, myoglobin, and κ-(BEDT-TTF)2Cu[N(CN)2]Br crystals. In the last section, we report the progress of the development of normal mode holography for soft materials. The new apparatus and scanning method is described, and results of the initial tests on the protein Photosystem II are discussed.

A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.

Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degreesC with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degreesC in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degreesC in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degreesC. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.

Human Hb, an alpha(2)beta(2) tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery, in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the a subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.

A comparison of the O-2 equilibrium curves of sperm-whale myoglobin locked in the liganded (CO-bound) and unliganded (deoxy) conformations by encapsulation in a wet porous sol-gel silica reveals a marked difference between them. The CO-bound state locked myoglobin showed a nearly monophasic (hyperbolic) O-2 equilibrium curve with a dissociation constant of 0.2 Torr, which is smaller than that of myoglobin in solution (0.5 Torr). On the other hand, the deoxy state-locked myoglobin exhibited a multiphasic O-2 equilibrium curve that can be represented by a sum of three independent components with dissociation constants of 0.19, 0.90, and 44 Torr, respectively, indicating that deoxymyoglobin exists in multiple conformations. These results show that myoglobin can be frozen into ligand-dependent conformational populations at room temperature in the wet sol-gel and suggest that the overall O-2 equilibrium properties of myoglobin in solution are generated by a redistribution of protein conformational populations in response to ligand binding.

Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug. The high resolution crystal structure of horse carbonmonoxyhemoglobin in complex with bezafibrate reveals that the bezafibrate molecule lies near the surface of the E-helix of each a subunit and the complex maintains the quaternary structure of the R-state. Binding is caused by the close fit of bezafibrate into the binding pocket, which is composed of some hydrophobic residues and the heme edge, suggesting the importance of hydrophobic interactions. Upon binding of bezafibrate, the distance between Fe and the Ne, of distal His E7(alpha58) is shortened by 0.22 Angstrom in the a subunit, whereas no significant structural changes are transmitted to the 13 subunit. Oxygen equilibrium studies of R-state-locked hemoglobin with bezafibrate in a wet porous sol-gel indicate that bezafibrate selectively lowers the oxygen affinity of one type of subunit within the R-state, consistent with the structural data. These results disclose a new allosteric mechanism of bezafibrate and offer the first demonstration of how the allosteric effector interacts with R-state hemoglobin.

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the (x heme, while it was similarly induced by binding of CO and NO to the,8 heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place In a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.

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, alpha(2)(Fe-CO)beta(2)(Ni) and alpha(2)(Ni)beta 2(Fe-CO), by encapsulation in the water-filled pores of sol-gel-derived transparent silica-gels. Ln our experimental system, nickel(II) protoporphyrin binds neither O-2 nor CO and mimics a fixed deoxyheme, and the gel matrix provides a means of inhibiting large-scale protein structural changes, thus enabling O-2 equilibrium study of the hybrids still in their doubly liganded conformations. Results showed that two conformations of widely different O-2 affinity exist together in each doubly liganded hemoglobin, providing a direct proof of the concerted mechanism versus the sequential mechanism. (C) 1999 Academic Press.

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.

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha(+CN-)beta(+CN-))(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad, Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha(+CN-)beta(+CN-))(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N-2 With HCN. This confirms our view that previous experimental data on (alpha(+CN-)beta(+CN-))(alpha beta) Obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha(+CN-)beta(+CN-))(alpha beta) (i.e., greater than or equal to -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha(+CN-)beta(+CN-))(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange, These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.

It has been reported that hybridization of the equimolar mixture of cyanomethemoglobin and deoxyhemoglobin through dimer exchange reaction results in establishment of an approximately binomial (1:2:1) equilibrium distribution of these parental hemoglobins and their hybrid molecule, (alpha(+CN-)beta(+CN-))-(alpha beta), within several days under anaerobic conditions at pH 7.4, 21.5 degrees C, leading to a hyper (i.e., about 170 times) thermodynamic stability of (alpha(+CN-)beta(+CN-))(alpha beta) relative to the stability of the other diliganded species at pH 7.4, 21.5 degrees C [Daugherty, M. A., Shea, M. A., Johnson, J. A., LiCata, V. J., Turner, G. J., & Ackers, G. K. (1991) Proc. Natl. Acad. Sci, U.S.A. 88, 1110-1114], To examine whether the published ''binomiality'' for this deoxy-cyanomet hybrid system really reflects the thermodynamic stability of (alpha(+CN-)-beta(+CN-))(alpha beta), we used a binomial. (1:2:1) equilibrium distribution of the equimolar mixture of cyanomethemoglobin and fully oxygenated hemoglobin as a starting condition, and then this system was rapidly deoxygenated. We found that the relative population of the hybrid was reduced to 8.6% of the total upon deoxygenation. It was also found that the hybridization experiment under anaerobic conditions was not allowed to continue for a long time due to a valency exchange reaction between deoxy and cyanomet derivatives. For instance, a 48 h incubation resulted in the oxidation of 44% of Fe2+ to Fe3+ hemes in the original deoxyhemoglobin and the reduction of 42% of Fe3+ to Fe2+ hemes in the original cyanomethemoglobin. These results suggest that a real distribution of the deoxy-cyanomet hybrid system at equilibrium is fairly far from 1:2:1 (binomial distribution), and the thermodynamic stability of (alpha(+CN-)beta(+CN-))(alpha beta) is less than one-tenth of the hyperstability previously reported. In addition, most of the previous results on deoxy-cyanomet valency hybrids placed under long anaerobic conditions should be subject to reexamination due to possible valency exchange reactions.

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. © 1995 Academic Press Limited.

We have previously reported that cross-linked asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha(Fe)beta(Fe)][alpha(Ni)beta(Ni)], in which the alpha 1 beta 1 dimer containing ferrous protoporphyrin IX and the adjacent alpha 2 beta 2 dimer containing nickel(II) protoporphyrin IX were cross-linked between Lys-82 beta(1) and Lys-82 beta(2) by reaction with bis(3,5-dibromosalicyl)fumarate, represents an adequate model for determination of the alpha 1 beta 1 oxygenation properties of native hemoglobin [Shibayama, N., Imai, K., Morimoto, H., and Saigo, S. (1993) Biochemistry 32, 8792-8798]. To extend the approach using cross-linked Ni(II)Fe(II) hybrids to all possible pathways for initial-half oxygenation of hemoglobin, we have prepared three other types of cross-linked Ni(II)-Fe(II) hybrids, carrying nickel(II) protoporphyrin IX in two subunits and ferrous protoporphyrin IX in the other two subunits, and have determined the two-step oxygen equilibrium curves of the ferrous subunits within these cross-linked hybrids. For the first step of oxygenation, the a subunit shows about 3-fold higher affinity than the beta subunit at all pH values examined, indicative of a significant functional heterogeneity of the subunits in deoxyhemoglobin. For the second step of oxygenation, the cooperativity represented by the Hill coefficient (n(max)) increases in the order of beta 1 beta 2 (n(max)= 1.36), alpha 1 beta 1 (n(max) = 1.41), alpha 1 beta 2 (n(max) = 1.64), and alpha 1 alpha 2 (n(max) = 1.72) at pH 7.4 in the presence of 0.1 M Cl- at 25 degrees C. In all hybrids, the oxygen affinities and cooperativities are pH-dependent, the Hill coefficients becoming larger as pH increases. These results imply that [alpha(Fe)beta(Fe-O-2)](2) is the only minor diliganded intermediate in the oxygen equilibrium of tetrameric hemoglobin. The implications of these findings for the mechanism of hemoglobin cooperativity are discussed.

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.

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.

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82-beta-1 and lysine-82-beta-2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93-beta-1 toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.