Subunit iron spin heterogeneity in human aquomethemoglobin A

On the basis of a reaction scheme in which the ligand binding steps are preceded by fast iron spin transitions (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334; Iwuoha, E.I. and Okonjo, K.O. (1985) Biochim. Biophys. Acta 829, 327-334), the spin equilibrium constants of methemoglobin subunits are calculated from kinetic and equilibrium binding parameters with azide ion as ligand. The results demonstrate the existence of thermodynamic spin heterogeneity within the tetramer.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

An effect of enzyme and ligand concentration on the state of aggregation of aspartate transcarbamylase of E. coli: I. The binding of CTP and ATP to the enzyme.

Detailed binding studies of the inhibitor, cytidine triphosphate (CTP), to native Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) reveal significant changes in subunit interaction when enzyme concentration is altered. In contrast, similar binding studies of the activator, adenosine triphosphate (ATP), do not reveal such changes, but do indicate more complex subunit interactions than previously reported. Equilibrium dialysis studies of 4 degrees C are consistent with six binding sites for CTP and ATP per enzyme molecule of molecular weight 310 000, at all enzyme concentrations. CTP binding studies reveal a progressive change from apparent positive to negative cooperativity as the enzyme concentration is decreased. ATP binding studies reveal complex subunit interactions involving a mixture of apparent negative and positive cooperativity. Sucrose gradient studies indicate the presence of at least three enzymatically active polymeric forms of the enzyme. The preliminary sedimentation studies indicate possible ligand and enzyme concentration perturbations of a preexisting association equilibrium in the aspartate transcarbamylase system. The binding data are therefore interpreted in terms of an association model.     

Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.     

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.     

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling

Structure-function relationships of the gamma-epsilon-c subunit interface of F(O)F(1) ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F(1) mutants from gammaLeu198 to gammaLeu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma201-209, which is reminiscent of a beta-strand. Labels at positions gamma204 and gamma210 show tertiary contact upon F(1) binding to F(O) and gammaD210C has reduced coupling efficiency. gammaE208C could not be spin labeled, but the uncoupling effects of gammaE208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-22297]. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma201 and gamma207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F(1) indicate that the interactions of these residues with the epsilon subunit mediate its inhibitory activity. Disrupted interactions with epsilon subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon-c interface of Escherichia coli F(O)F(1) ATP synthase in which side chains from the odd-numbered residues of the gammaLys201-gammaTyr207 beta-strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gammaAsp204, gammaGlu208, and gammaAsp210 interact with the F(O) sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.     

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.     

ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli

The tau and gamma subunits of the DNA polymerase III holoenzyme of Escherichia coli were each isolated in large quantities as oligomers from overproducing cells in which their genes (dnaZ and X) were under the control of a T7 phage promoter. The 52-kDa gamma subunit (encoded by the dnaZ sequence) contains three-forths of the N-terminal residues of the 71-kDa tau subunit (encoded by the dnaX sequence). Both gamma and tau share a binding site for ATP (or dATP). A DNA-dependent ATPase activity (Lee, S.H., and Walker, J.R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2713-2717) exhibited only by the tau subunit, presumably requires a DNA-binding site in the C-terminal domain lacking in the gamma subunit. Among ATPases dependent on single-stranded DNA, the tau activity is remarkable in the failure of homopolymers (e.g. poly(dA) or poly(dT)) to replace natural DNAs. The presumed need for certain secondary structures may reflect a feature of template binding in the crucial contribution that tau makes to the high processivity of polymerase III holoenzyme. Limited tryptic digestion of tau generates a fragment that resembles gamma in: (i) size, (ii) binding of ATP without ATPase activity, and (iii) a level of complementing holoenzyme activity in extracts of dnaZ-mutant cells that is higher than that of tau.     

The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy.

The interaction forces between biotin and a set of streptavidin site-directed mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force between the site-directed (Trp to Phe or Ala) mutants. The interaction force is poorly correlated with both the equilibrium free energy of biotin binding and the activation free energy barrier to dissociation of the biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as the enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force is clearly correlated with the activation enthalpy of dissociation. These results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.     

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.     

A re-examination of the cleavage of fibrinogen and fibrin by plasmin.

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000 to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from its gamma' subunit of 38,000 molecular weight; however, the beta' subunit of 42,000 molecular weight and the alpha' subunit of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.     

Characterization of the gamma subunits of the 7S nerve growth factor complex.

The gamma subunits of the 7S nerve growth factor complex (7S NGF) display arginine esteropeptidase activity. By varying the conditions of electrophoresis in acrylamide gel, it has been demonstrated that the gamma-subunit fraction of 7S NGF contains five different proteins, in contrast to the three (gamma1, gamma2, and gamma3) originally described (Smith, A.P., Varon, S. and Shooter, E.M. (1968), Biochemistry 7, 3259-3268); the gamma1 and gamma2 subunits, previously thought to be single species, can each be resolved into two components. The two components of the gamma1 subunit have the same isoelectric point, as do the two components of the gamma2 subunit. The distribution of protein among the two components of each of the gamma1 and gamma2 subunits varied from preparation to preparation. Moreover, a shift in the distribution for the gamma1 subunit was accompanied by a parallel shift for the gamma2 subunit. All of the different gamma proteins have the same molecular weight. On the basis of the molecular weights of the peptide chains of the gamma subunits and of the species which are formed by cross-linking with dimethyl suberimidate, it was concluded, that both the gamma1 and gamma2 subunits contain one species with two peptide chains and another with three peptide chains, while the gamma3 subunit is a single species with three peptide chains. The results also suggest that two of the chains in the three-chain species are derived, by proteolytic cleavage, from the larger chain in the two-chain species.     

Gbetagamma affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the gamma subunit

Two native betagamma dimers, beta(1)gamma(1) and beta(1)gamma(2), display very different affinities for receptors. Since these gamma subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gbetagamma interaction with receptors. We constructed baculoviruses encoding gamma(1) and gamma(2) subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the gamma chains and a set of gamma(1) and gamma(2) chimeras with the gamma(2) CAAX motif at the carboxyl terminus. All the gamma constructs coexpressed with beta(1) in Sf9 cells yielded beta(1)gamma dimers, which were purified to near homogeneity, and their affinities for receptors and Galpha were quantitatively determined. Whereas alteration of the isoprenoid of gamma(1) from farnesyl to geranylgeranyl and of gamma(2) from geranylgeranyl to farnesyl had no impact on the affinities of beta(1)gamma dimers for Galpha(t), the non-prenylated beta(1)gamma(2) dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the beta(1)gamma(2) dimer for rhodopsin and a <3-fold change for the beta(1)gamma(1) dimer. In each case with identical isoprenylation, the beta(1)gamma(2) dimer displayed significantly greater affinity for rhodopsin compared with the beta(1)gamma(1) dimer. Furthermore, dimers containing chimeric Ggamma chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Ggamma subunit is essential for binding to both Galpha and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Galpha. Finally, the primary structure of the Ggamma subunit provides a major contribution to receptor binding of Gbetagamma, with the carboxyl-terminal sequence conferring receptor selectivity.     

Does a monospecific hybridoma always secrete homogeneous immunoglobulins?

The fusion of splenocytes (from mice immunized with the beta 2 subunit of E. coli tryptophan synthase) with myeloma cells which do not produce immunoglobulins gave rise to a clone secreting immunoglobulins with two distinct isotypes : gamma 1 and gamma 2b (Djavadi-Ohaniance et al. (1984) Biochemistry, 23, 97-104). Analysis of the immunoglobulins secreted by this clone indicates that these two isotypes are carried by two distinct heavy chains which are able to randomly associate to form hybrid molecules. In addition, two classes of light chains are able to randomly and to form heterologous associations with both the gamma 1 and gamma 2b heavy chains. Only the association between the gamma 2b heavy chains with one of the two classes of light chains leads to a combining site specific for the binding of the antigen beta 2.