Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.     

Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.     

Ligand binding to heme proteins. VI. Interconversion of taxonomic substates in carbonmonoxymyoglobin.

The kinetic properties of the three taxonomic A substates of sperm whale carbonmonoxy myoglobin in 75% glycerol/buffer are studied by flash photolysis with monitoring in the infrared stretch bands of bound CO at nu(A0) approximately 1967 cm-1, nu(A1) approximately 1947 cm-1, and nu(A3) approximately 1929 cm-1 between 60 and 300 K. Below 160 K the photodissociated CO rebinds from the heme pocket, no interconversion among the A substates is observed, and rebinding in each A substate is nonexponential in time and described by a different temperature-independent distribution of enthalpy barriers with a different preexponential. Measurements in the electronic bands, e.g., the Soret, contain contributions of all three A substates and can, therefore, be only approximately modeled with a single enthalpy distribution and a single preexponential. The bond formation step at the heme is fastest for the A0 substate, intermediate for the A1 substate, and slowest for A3. Rebinding between 200 and 300 K displays several processes, including geminate rebinding, rebinding after ligand escape to the solvent, and interconversion among the A substates. Different kinetics are measured in each of the A bands for times shorter than the characteristic time of fluctuations among the A substates. At longer times, fluctuational averaging yields the same kinetics in all three A substates. The interconversion rates between A1 and A3 are determined from the time when the scaled kinetic traces of the two substates merge. Fluctuations between A1 and A3 are much faster than those between A0 and either A1 or A3, so A1 and A3 appear as one kinetic species in the exchange with A0. The maximum-entropy method is used to extract the distribution of rate coefficients for the interconversion process A0 <--> A1 + A3 from the flash photolysis data. The temperature dependencies of the A substate interconversion processes are fitted with a non-Arrhenius expression similar to that used to describe relaxation processes in glasses. At 300 K the interconversion time for A0 <--> A1 + A3 is 10 microseconds, and extrapolation yields approximately 1 ns for A1 <--> A3. The pronounced kinetic differences imply different structural rearrangements. Crystallographic data support this conclusion: They show that formation of the A0 substate involves a major change of the protein structure; the distal histidine rotates about the C(alpha)-C(beta) bond, and its imidazole sidechain swings out of the heme pocket into the solvent, whereas it remains in the heme pocket in the A1 <--> A3 interconversion. The fast A1 <--> A3 exchange is inconsistent with structural models that involve differences in the protonation between A1 and A3.     

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.     

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational effects at the subunit interfaces of human hemoglobin: evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the alpha 1 beta 2 interface

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of beta Y145, the residues with the most significant effects on these kinetics are found in the hinge region of the alpha 1 beta 2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both alpha or both beta subunits, were prepared for beta W37E, beta W37A, alpha Y140G, and alpha Y140A, hinge region variants causing large kinetic changes, and for beta Y145G. Such hybrids permit measurements of the kinetics of CO binding to only the heme-containing alpha or beta subunits within the unliganded hemoglobin tetramer. Mutations at beta 37 and alpha 140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of beta Y145 has a large effect on the beta subunits in the deoxygenated T state, but very little effect on the alpha subunits. Oxygen equilibria measurements on the crystalline T state of beta W37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the alpha 1 beta 2 interface.     

Allosteric kinetics and equilibria of triligated, cross-linked hemoglobin.

Using modulated excitation, we have measured the forward and reverse rates of the allosteric transition between relaxed (R) and tense (T) quaternary structures for triply ligated hemoglobin (Hb), cross-linked between the alpha chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris buffers at neutral pH. Since the cross-link prohibits disproportionation, triply ligated aquomet Hb species with ferrous beta chains were specifically isolated by isoelectric focusing. Modulated excitation provides rate pairs and therefore gives equilibrium constants between quaternary structures. To coordinate with that information, oxygen binding curves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is determined by modulated excitation to be of order unity for O2 or CO (1.1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subunits it is much greater (> or = 23). R-->T conversion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from HbXL O2 were used to obtain a unique allosteric decomposition of the ferrous O2 binding curve in terms of KT, KR, and L3. From these values and the O2 binding curve of tri-aquomet HbXL, L3 was calculated to be 2.7 for the tri-aquomet derivative. Consistency in L3 values between equilibrium and modulated excitation data for tri-aquomet-HbXL can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than that for binding to the beta chains in the R state, while the cooperative properties remain homogeneous. The results are in quantitative agreement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the alpha subunits with almost no change in the affinity of the beta subunits, leaving the allosteric parameters L and c unchanged.     

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.     

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.     

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.     

Interaction of fully liganded valency hybrid hemoglobin with inositol hexaphosphate. Implication of the IHP-induced T state of human adult methemoglobin in the low-spin state

To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A.     

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: NMR studies of their tertiary and quaternary structures

Diruthenium-substituted Ru-Fe hybrid hemoglobins (Hb) were synthesized by heme substitution from protoheme to ruthenium (II) carbonyldeuteroporphyrin in the alpha or beta subunits. As the carbon monoxide coordinated to ruthenium (II) is not released under physiological conditions, deoxygenated Ru-Fe hybrid derivatives [alpha(Fe)2 beta(Ru-CO)2 and alpha(Ru-CO)2 beta(Fe)2] can serve as models for half-liganded Hbs. On the basis of proton NMR spectra of hyperfine-shifted proton resonances, these Ru-Fe hybrid Hbs have only small structural changes in the heme environment of the partner subunits at low pH. The proton NMR spectra of the intersubunit hydrogen-bonded protons also showed that the quaternary structures of the two complementary hybrids both remain in the "T-like state" at low pH, suggesting that the T to R structural conversion is induced by ligation of the third ligand molecule. Marked conformational changes in the heme vicinity are observed at high pH only for alpha(Ru-CO)2 beta(Fe)2, and its quaternary structure is converted into the "R state"; the alpha(Fe)2 beta(Ru-CO)2 hybrid does not undergo this change. This implies that the free-energy difference between the two quaternary states is smaller in the alpha-liganded hybrid than in the beta-liganded one.     

Monooxygenase activity of human hemoglobin: NMR demonstration of different modes of substrate binding corresponding to different activities of hemoglobin derivatives

In the accompanying paper [Ferraiolo, B. L., Onady, G. M., & Mieyal, J. J. (1984) Biochemistry (preceding paper in this issue)] we reported different aniline hydroxylase activities for ferrihemoglobin, its isolated subunits, and the converse pair of valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 in a reconstituted system containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and cytochrome P-450 reductase. To investigate the molecular basis for the different activities, 1H NMR T1 relaxation studies of aniline were performed in the absence and presence of each of the hemoglobin (Hb) species. The paramagnetic contribution of the ferric heme iron atoms of each Hb derivative to the enhanced relaxation of the proton nuclei of aniline was determined relative to control experiments in which the hemoproteins had been converted fully to the corresponding (carbonmonoxy)ferrous forms, which are diamagnetic. According to the known distance dependence of the paramagnetic effect and the relative changes in T1 for the upfield and downfield signals in the spectrum of aniline, it was ascertained that aniline binds in the same manner to the beta-ferric hybrid and to ferrihemoglobin. These two forms displayed equivalent hydroxylase activities that were the highest among the Hb derivatives for the same aniline concentration. The T1 changes observed with the alpha-ferric hybrid suggest a different orientation for aniline in that complex. The T1 data for the isolated subunits alpha 3+ and beta 3+4 would indicate that overall binding of aniline includes a component of direct aniline-heme ligation in each case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical studies of DNA-RNA hybrid conformations.

Molecular modelling has been used to probe the conformational preferences of double stranded DNA-RNA hybrids. As might be expected, the sugars of the DNA strand have higher conformational flexibility, but, for the majority of the repetitive sequences studied, these sugars prefer a C2-endo pucker, while ribose sugars uniformly adopt a C3-endo pucker. This gives rise to a strongly heteronomous duplex conformation. One exception to this rule involves the thymidine strand of poly(dT).poly(rA), which marginally prefers a C3-endo pucker. Our study further indicates that the DNA strands of the hybrids favour backbone torsions in the canonical B domain, rather than the modified values proposed on the basis of fibre diffraction studies. Backbone conformational transitions can nevertheless be induced leading to an alpha gamma-flip (alpha:gamma, g-/g(+)-->t/t) or to the alpha beta gamma-flip form proposed from fibre studies (alpha:beta:gamma, g-/t/g(+)-->t/g+/t). The latter transition is also found to be linked to BI-->BII transitions (epsilon:zeta, t/g(-)-->g-/t).     

Double-mixing kinetic studies of the reactions of methyl isocyanide and CO with diliganded intermediates of hemoglobin: alpha 2CO beta 2 and alpha 2 beta 2CO

Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.     

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.