Mortality risk factors and fulminant sub-phenotype in anaerobic bacteremia: a 10-year retrospective, multicenter, observational cohort study.

PURPOSE: During the last decade, the incidence of anaerobic bacteremia (AB) has been increasing. Patients with AB may develop complex underlying diseases, which can occasionally be accompanied by fatal or fulminant outcomes. However, the risk factors for AB-related mortality remain unclear. Herein, we sought to elucidate the risk factors for AB-related mortality. METHODS: In this multicenter, retrospective, observational study, we enrolled patients with culture-proven AB from six tertiary hospitals in Japan, between January 2012 and December 2021. Data on patient and infection characteristics, laboratory findings, treatment, and outcome were collected, and their associations with mortality were analyzed. RESULTS: A total of 520 participants were included. The 30-day mortality in the study cohort was 14.0% (73 patients), and malignant tumors were frequently observed comorbidities in 48% of the entire cohort. Multivariable logistic regression analysis showed a Charlson comorbidity score of > 6, serum creatinine level of > 1.17 mg/dL, and hypotension to be independent risk factors for 30-day mortality in AB (odds ratios [ORs] 2.12, 2.25, and 5.12, respectively; p < 0.05), whereas drainage significantly reduced this risk (OR, 0.28; p < 0.0001). Twelve patients (2.3% of the whole cohort and 16.4% of the deceased patients) presented with extremely rapid progression leading to fatal outcome, consistent with "fulminant AB." CONCLUSIONS: This study identified acute circulatory dysfunction and performance of drainage as independent predictive factors for 30-day AB-related mortality and revealed the existence of a fulminant AB sub-phenotype. Our findings could serve as a practical guide to predict the clinical outcomes of AB.

Preliminary time-of-flight neutron diffraction study of human deoxyhemoglobin.

Human hemoglobin (HbA) is an intricate system that has evolved to efficiently transport oxygen molecules (O(2)) from lung to tissue. Its quaternary structure can fluctuate between two conformations, T (tense or deoxy) and R (relaxed or oxy), which have low and high affinity for O(2), respectively. The binding of O(2) to the heme sites of HbA is regulated by protons and by inorganic anions. In order to investigate the role of the protonation states of protein residues in O(2) binding, large crystals of deoxy HbA (approximately 20 mm(3)) were grown in D(2)O under anaerobic conditions for neutron diffraction studies. A time-of-flight neutron data set was collected to 1.8 A resolution on the Protein Crystallography Station (PCS) at the spallation source run by Los Alamos Neutron Science Center (LANSCE). The HbA tetramer (64.6 kDa; 574 residues excluding the four heme groups) occupies the largest asymmetric unit (space group P2(1)) from which a high-resolution neutron data set has been collected to date.

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution.

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T:-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S:-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15-50 degrees C, the S:-form the second and the T:-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.

Polymerization and solubility of Ni(II)-Fe(II) hybrid Hb S.

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 2s (Fe-CO) and alpha 2 (Fe-CO) beta 2s (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 2s (Fe-CO) and alpha 2 (Fe-CO) beta 2s (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.

X-ray absorption spectroscopic studies of a transient intermediate in the reaction of cyanide metmyoglobin with dithionite by using rapid freezing.

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 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.

Functional analysis of hemoglobin molecules locked in doubly liganded conformations.

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, alpha2(Fe-CO)beta2(Ni) and alpha2(Ni)beta2(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.

Structure of haemoglobin in the deoxy quaternary state with ligand bound at the alpha haems.

We report the X-ray crystal structure of two analogues of human haemoglobin in the deoxy quaternary (T) state with ligand bound exclusively at the alpha haems. These models were prepared from symmetric, mixed-metal hybrid haemoglobin molecules. The structures of alpha Fe(II) beta Co(II), its carbonmonoxy derivative alpha Fe(II)CO beta Co(II), and alpha Fe(II)O2 beta Ni(II) are compared with native deoxy haemoglobin by difference Fourier syntheses at 2.8, 2.9 and 3.5 A resolution, respectively, and the refined alpha Fe(II)CO beta Co(II) structure is analysed. In both the native deoxy and liganded T molecules, the mean plane of the alpha-subunit haem is parallel with the axis of the F helix, but this plane is tilted with respect to the helix axis in the oxy-quaternary R state. The side-chains of LeuFG3 and ValFG5 sterically restrict haem tilting in the T state. We propose that strain energy develops at the contact between the haem and these residues in the liganded T-state haemoglobin, and that the strain is, in part, responsible for the low affinity of the T-state alpha haem.

Fixation of the quaternary structures of human adult haemoglobin by encapsulation in transparent porous silica gels.

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.

Properties of chemically modified Ni(II)-Fe(II) hybrid hemoglobins. Ni(II) protoporphyrin IX as a model for a permanent deoxy-heme.

Chemical modifications, NES-Cys(beta 93), des-Arg(alpha 141), and both modifications on the same molecule, were made to Ni-Fe hybrid hemoglobins, and their effect on individual subunits was investigated by measuring oxygen equilibrium curves, the Fe(II)-N epsilon (His F8) stretching Raman lines, and light-absorption spectra. The oxygen equilibrium properties indicated that modified Ni-Fe hybrid hemoglobins remain good models for the corresponding deoxy ferrous hemoglobins, although K1, the dissociation equilibrium constant for the first oxygen to bind to hemoglobin, was decreased by the chemical modifications. Resonance Raman spectra of deoxy alpha 2 (Fe) beta 2 (Ni) and light-absorption spectra of deoxy alpha 2 (Ni) beta 2 (Fe), revealed that the state of alpha hemes in both hybrid hemoglobins underwent a transition from a deoxy-like state to an oxy-like state caused by these chemical modifications when K1 was about 3 mm Hg (1 mm Hg approximately 133.3 Pa). On the other hand, the state of beta hemes in hybrid hemoglobins was little affected, when K1 was larger than 1 mm Hg. Modified alpha 2 (Fe) beta 2 (Ni) gave a Hill coefficient greater than unity with a maximum of 1.4 when K1 was about 4 mm Hg. The two-state model predicts that the K1 value at the maximum Hill coefficient should be much larger than this value. For oxygen binding to unmodified alpha 2 (Ni) beta 2 (Fe), oxygen equilibrium data suggested no structural change, while the spectral data showed a structural change around Ni(II) protoporphyrin IX in the alpha subunits. A similar situation was encountered with modified alpha 2 (Ni) beta 2 (Fe), although K1 was decreased as a result of the structural changes induced by the modifications.

Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins.

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.

Structure of deoxy-quaternary haemoglobin with liganded beta subunits.

We have determined the structure of a T-state haemoglobin in which the haem groups of the beta subunits have carbon monoxide bound, and the alpha subunits have nickel replacing the haem iron and are ligand-free. The structural adjustments on binding ligand in the T state are in the same direction as those associated with the quaternary transition, and a translational shift of the haem is severely restricted. We explain how these observations may account for the low ligand affinity of the beta haem of T-state haemoglobin.

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(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 alpha2(Fe)beta2(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 effectors, 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 alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(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 (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors 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.

Direct observation of two distinct affinity conformations in the T state human deoxyhemoglobin.

The main features of cooperative oxygenation of 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.

Oxygen equilibrium and electron paramagnetic resonance studies on copper(II)-iron(II) hybrid hemoglobins at room temperature.

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.

Development of serum IgM antibodies against superantigens of Staphylococcus aureus and Streptococcus pyogenes in Kawasaki disease.

To serologically determine the association of microbial superantigens and the pathogenesis of Kawasaki disease (KD), we conducted a case-control study. Serum IgG and IgM antibodies against staphylococcal enterotoxin A (SEA), SEB, SEC, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxin A (SPEA) were measured by an enzyme-linked immunosorbent assay in 293 serum samples from 65 KD patients on clinical days 1-28 and 120 control samples. The administration of immunoglobulin products, which contain high concentrations of IgG antibodies against all the superantigens, directly elevated antitoxin IgG antibodies in KD patients. In contrast, antitoxin IgM antibodies were not detected in immunoglobulin products. Actually, we found a significant elevation of IgM antibodies against SEA in KD patients in the first (median titre: 0.020, P < 0.01 versus control), second (0.024, P < 0.001), third (0.030, P < 0.001) and fourth (0.038, P < 0.001) weeks, compared to the controls (0.015). Significant differences of IgM antibodies were also true for SEB, TSST-1, and SPEA throughout the first to fourth weeks, and for SEC throughout the second to fourth weeks. The prevalence of KD patients having high IgM titres (> mean + 2SD of control values) to the 5 superantigens was increased with the clinical weeks, and reached 29-43% of KD subjects at the fourth week. This is the first study that describes kinetics of IgM antibodies against superantigens and clarifies the serological significance throughout the clinical course of KD. Our results suggest that multiple superantigens involve in the pathogenesis of KD.

Reexamination of the hyper thermodynamic stability of asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): no preferentially populating asymmetric hybrid at equilibrium.

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.

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange.

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 N2 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., >/= -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.

Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)-iron(II) hybrid hemoglobins.

Ni(II)-Fe(II) hybrid hemoglobins, alpha(Fe)2 beta(Ni)2 and alpha(Ni)2 beta(Fe)2 have been characterized by proton nuclear magnetic resonance with Ni(II) protoporphyrin IX (Ni-PP) incorporated in apoprotein, which serves as a permanent deoxyheme. alpha(Fe)2 beta(Ni)2, alpha(Ni)2 beta(Fe)2, and NiHb commonly show exchangeable proton resonances at 11 and 14 ppm, due to hydrogen-bonded protons in a deoxy-like structure. Upon binding of carbon monoxide (CO) to alpha(Fe)2 beta(Ni)2, these resonances disappear at pH 6.5 to pH 8.5. On the other hand, the complementary hybrid alpha(Ni)2 beta(Fe-CO)2 showed the 11 and 14 ppm resonances at low pH. Upon raising pH, the intensities of both resonances are reduced, although these changes are not synchronized. Electronic absorption spectra and hyperfine-shifted proton resonances indicate that the ligation of CO in the beta(Fe) subunits induced changes in the coordination and spin states of Ni-PP in the alpha subunits. In a deoxy-like structure, the coordination of Ni-PP in the alpha subunits is predominantly in a low-spin (S = 0) four-coordination state, whereas in an oxy-like structure the contribution of a high-spin (S = 1) five-coordination state markedly increased. Ni-PP in the beta subunits always takes a high-spin five-coordination state regardless of solution conditions and the state of ligation in the partner alpha(Fe) subunits. In the beta(Ni) subunits, a significant downfield shift of the proximal histidyl N delta H resonance and a change in the absorption spectrum of Ni-PP were detected, upon changing the quaternary structure of the hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen equilibrium properties of asymmetric nickel(II)-iron(II) hybrid hemoglobin.

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 complementary alpha 2 beta 2 dimer containing Ni(II) protoporphyrin IX were cross-linked between Lys-82 beta 1 and Lys-82 beta 2 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 alpha 1 beta 1 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 alpha 2 subunit was changed upon carbon monoxide (or oxygen) binding to the alpha 1 beta 1 dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen equilibrium properties of nickel(II)-iron(II) hybrid hemoglobins cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl)fumarate: determination of the first two-step microscopic Adair constants for human hemoglobin.

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., & 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 alpha 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 (nmax) increases in the order of beta 1 beta 2 (nmax = 1.36), alpha 1 beta 1 (nmax = 1.41), alpha 1 beta 2 (nmax = 1.64), and alpha 1 alpha 2 (nmax = 1.72) at pH 7.4 in the presence of 0.1 M Cl- at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)