Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.     

The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.     

UV resonance raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin.

We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.     

Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels

Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm(-1). The low and high values correspond to the initial deoxy T state and liganded R (R(2)) state species, respectively. The 222 and 227 cm(-1) species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the alpha(1)beta(2) interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.     

Beta 93 modified hemoglobin: kinetic and conformational consequences.

The reactive sulfhydryl on Cys beta93 in human adult hemoglobin (HbA) has been the focus of much attention. It has purported functional roles such as a transporter of nitric oxide and a detoxifier of super oxide. In addition, it has a proposed role in the allosteric mechanism. The present study addresses the functional and conformational consequences of modifying the beta93 sulfhydryl using either maleimide or disulfide-based reactions. The geminate and bimolecular recombination of CO derivatives of several different beta93-modified Hbs in both solution and sol-gel matrixes provide a window into functional modifications associated with both the R and T states of these proteins. Nanosecond time-resolved visible resonance Raman spectroscopy is used to probe conformational consequences associated with the proximal heme environment. The results show functional and conformational consequences that depend on the specific chemistry used to modify beta93. Maleimide-based modification show the most significant alterations of R-state properties including a consistent pattern of a reduced geminate yield and a loss of the favorable heme-proximal histidine interaction normally seen for liganded R-state HbA. A mechanism based on a displacement of the side chain of Tyr beta145 is explored as a basis for this effect as well as other situations where there is loss of the quaternary enhancement effect. The quaternary enhancement effect refers to the enhancement of ligand binding properties of the alphabeta dimers when they are associated into the R-state tetramer.     

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.     

Nitrite reductase activity of sol-gel-encapsulated deoxyhemoglobin. Influence of quaternary and tertiary structure

Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.     

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.     

Molecular dynamics simulations of hemoglobin A in different states and bound to DPG: effector-linked perturbation of tertiary conformations and HbA concerted dynamics

Recent functional studies reported on human adult hemoglobin (HbA) show that heterotropic effector-linked tertiary structural changes are primarily responsible for modulating the oxygen affinity of hemoglobin. We present the results of 6-ns molecular dynamics simulations performed to gain insights into the dynamical and structural details of these effector-linked tertiary changes. All-atom simulations were carried out on a series of models generated for T- and R-state HbA, and for 2,3-diphosphoglycerate-bound models. Cross-correlation analyses identify both intra- and intersubunit correlated motions that are perturbed by the presence of the effector. Principal components analysis was used to decompose the covariance matrix extracted from the simulations and reconstruct the trajectories along the principal coordinates representative of functionally important collective motions. It is found that HbA in both quaternary states exists as ensembles of tertiary conformations that introduce dynamic heterogeneity in the protein. 2,3-Diphosphoglycerate induces significant perturbations in the fluctuations of both HbA states that translate into the protein visiting different tertiary conformations within each quaternary state. The analysis reveals that the presence of the effector affects the most important components of HbA motions and that heterotropic effectors modify the overall dynamics of the quaternary equilibrium via tertiary changes occurring in regions where conserved functionally significant residues are located, namely in the loop regions between helices C and E, E and F, and F and G, and in concerted helix motions. The changes are not apparent when comparing the available x-ray crystal structures in the presence and absence of effector, but are striking when comparing the respective dynamic tertiary conformations of the R and T tetramers.     

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

Alteration of T-state binding properties of naturally glycated hemoglobin, HbA1c

The thermodynamic and kinetic properties of the most abundant glycated hemoglobin in human blood, HbA1c, have been studied in detail. They display significant differences as compared to normal hemoglobin, HbA0, in that (1) the shape of the oxygen binding curve of HbA1c in the Hill plot is markedly asymmetrical, with a lower asymptote extending up to approximately 40% oxygen saturation, and the oxygen affinity of the T state being tenfold higher than in HbA0; (2) oxygen pulse experiments on HbA1c show a slower rate of ligand dissociation (k = 25 s-1) even at low levels of oxygen saturation, where the T state is largely predominant; (3) kinetics of CO combination to deoxy HbA1c followed by means of stopped-flow experiments reveal the presence of a quickly reacting component, whose fraction increases upon dilution of hemoglobin. These results show that in contrast to what has been stated by other authors, HbA1c displays functional properties markedly different from HbA0. Analysis indicates that glycation of human hemoglobin affects the T quaternary structure, bringing about a more "relaxed" T state and leading to preferential binding to one type of chain (which is unaffected by chloride ions).     

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.     

Quaternary-transformation-induced changes at the heme in deoxyhemoglobins

Quaternary-structure-induced differences in both the high- and low-frequency regions of the resonance Raman spectrum of the heme have been detected in a variety of hemoglobins. These differences may be the result of (1) changes in the amino acid sequence, induced by genetic and chemical modifications, and (2) alterations in the quaternary structure. For samples in solution in low ionic strength buffers, differences in the 1357-cm-1 line (an electron-density-sensitive vibrational mode) correlate with differences in the 216-cm-1 line (the iron-histidine stretching mode). Thus, changes in the iron-histidine bond and changes in the pi-electron density of the porphyrin depend upon a common heme-globin interaction. The quaternary-structure-induced changes in the vibrational modes associated with the heme demonstrate that there is extensive communication between the heme and the globin and impact on models for the energetics of cooperativity. The local interactions of the iron-histidine mode are energetically small and destabilize the deoxy heme in the T structure with respect to the R structure. Therefore, these interactions must be larger in the ligated protein than in the deoxy protein to obtain a negative free energy of cooperativity. Additionally, our data imply that the deprotonation of the proximal histidine does not play a major role in the energetics of cooperativity. On the other hand, models for cooperativity that require conformational changes in the iron-histidine bond or direct interaction between the porphyrin and the protein are qualitatively consistent with the observed variation of heme electronic structure in concert with protein quaternary structure.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Study of the conversion of GDP-mannose into GDP-fucose in Nereids: a biochemical marker of oocyte maturation

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.     

Functional comparison of specifically cross-linked hemoglobins biased toward the R and T states.

Selected functional and spectroscopic properties of two human hemoglobin (HbA0) derivatives that were site-specifically cross-linked in the cleft between beta-chains where 2, 3-bisphosphoglycerate normally binds have been determined to assess the effects of the cross-linking on the behavior of the protein. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) cross-links Hb between beta82Lys residues. The resulting TTDS-Hb exhibits a slower rate of oxygen dissociation and an increased rate of carbon monoxide association than observed for HbA0. The electron paramagnetic resonance (EPR) spectrum of TTDS-HbNO does not exhibit the hyperfine structure that is indicative of significant conformational change despite the fact that the 2,3-bisphosphoglycerate binding site is occupied by the cross-linking reagent. The reactivity of the beta93Cys residues of TTDS-Hb is only slightly decreased relative to that of HbA0. On the other hand, cross-linking Hb between Lys82 and the amino-terminal beta1Val group with trimesoyl tris(methyl phosphate) (TMMP) increases the rate of oxygen dissociation and reduces the rate of CO association relative to the rates observed for HbA0. In addition, the EPR spectrum of the TMMP-HbNO exhibits the three-line hyperfine structure that results from disruption of the proximal His-Fe bond of the alpha-chains, and the accessibility of the betaCys93 residues in this derivative is decreased fourfold. The present results are consistent with the conclusion that the quaternary structure of TTDS-Hb is shifted toward the R state whereas the quaternary structure of TMMP-Hb is shifted toward the T state and provides additional evidence that the identity of the residues involved in intramolecular cross-linking of hemoglobin within the 2,3-bisphosphoglycerate binding site between beta-chains can have a significant influence on the conformational and functional properties of the protein.     

A photolysis-triggered heme ligand switch in H93G myoglobin

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.