Ligand binding properties of bacterial hemoglobins and flavohemoglobins

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Ligand binding properties of cytochromes c'.

The cytochromes c' bind CO, alkylisocyanides and CN- with rate and equilibrium constants which are 10(2)- to 10(6)-fold smaller than other high-spin hemoproteins. The decreased affinity for exogenous ligands is largely associated with steric interactions at the heme coordination site. While CO and alkylisocyanides bind noncooperatively to the dimeric Rhodospirillum molischianum cytochrome c', CO, alkylisocyanides and CN- appear to bind cooperatively to the dimeric Chromatium vinosum cytochrome c' due to a ligand-linked dimer-monomer dissociation equilibrium. The differences between the cytochromes c' are thought to be due to differences in amino acid residues near the heme coordination site and subunit interface.     

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.     

Ligand binding properties of human galanin receptors

Abstract

The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.     

Resonance Raman studies of hemoglobins reconstituted with mesoheme. Unperturbed iron-histidine stretching frequencies in a functionally altered hemoglobin

Hybrid hemoglobins, containing mesoheme in one type of subunit and protoheme in the partner subunits, have been studied by resonance Raman spectroscopy. These hybrids have been studied in both the met hybrid and fully reduced, deoxy forms. Judicious choice of laser excitation frequency permits selective enhancement of modes associated with each type of subunit; i.e., either meso- or protoheme-containing subunit. The assignments of low-frequency modes of meso- and protoheme are briefly discussed with special reference to the iron-histidine linkage. Despite functional differences between the hybrids, no significant changes in the strength of the iron-histidine linkages are detected by resonance Raman spectroscopy. These results are discussed with reference to recent high-resolution NMR studies of these same hybrids.     

3(17)beta-Hydroxysteroid Dehydrogenase of Pseudomonas testosteroni. Ligand binding properties.

The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.     

Synthesis and protein binding properties of T-antigen containing GlycoPAMAM dendrimers.

Allyl O-(beta-D-galactopyranosyl)-(1-3)-2-acetamido-2-deoxy-alpha-D-galactopyranoside (8) was prepared in excellent yield from the corresponding galactosyl bromide (6, 7) and allyl 2-acetamido-4,6-benzylidene-2-deoxy-alpha-D-galactopyranoside (5) using Hg(CN)2 as a promoter. Compound 5 was obtained from N-acetylglucosamine 1 following sequential protecting group strategy and C-4 epimerization as a key step. Carboxylic acid functionalized T-antigen derivative 15, obtained by radical addition of 3-mercaptopropionic acid to allyl disaccharide 10, was conjugated to PAMAM dendritic cores 13-16 by an efficient amide coupling strategy using TBTU. GlycoPAMAM dendrimers having T-antigen residues with 4, 8, 16 and 32 valencies (17-20) were obtained in 73 to 99% yields. Their protein binding properties were demonstrated using peanut lectin from Arachis hypogaea and a mouse monoclonal IgG antibody. The higher valency conjugates generated stronger binding interactions indicating a cluster effect. The inhibitory potential of these glycoPAMAM conjugates toward antibody-coating antigen interactions was enhanced up to 3800 times over that of the monomeric T-antigen residue (10).     

Ligand binding and detoxication

In an organism the binding of a toxic chemical to a binding site can act as a detoxication mechanism when toxicity is a property of the unbound ligand. This qualitative statement has been evaluated in quantitative terms. To this end parameters have been defined for which numerical values are required, equations are derived and a procedure is outlined that allows assessment of when and to what extent binding is of value in detoxication. In the process two new quantities are introduced, i.e. the binding capacity and the binding activity, which make for easier handling and comparison of binding data. It is concluded that to be important in detoxication the numerical value of the binding activity must be greater than unity and the total ligand concentration should not exceed the binding capacity. These general conclusions can be further refined depending on the accuracy with which the values of the parameters involved are known. Due to its generality the results of the analysis are useful in all situations where it is desirable to know the magnitude of the free fraction of a bound chemical.     

Ligand binding and self-association of phosphorylase b.

The mutual influence of ligand binding and self-association has been examined for phosphorylase b in the presence of a series of small ligands. The stepwise equilibrium constants describing the mutual dependence have been evaluated and discussed in terms of possible molecular mechanisms.     

Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes.

The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.     

Spectroscopic properties of the nitric oxide derivative of ferrous man, horse, and ruminant hemoglobins: a comparative study.

The spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous man, horse, buffalo, deer, mouflon, musk ox, ox, and reindeer hemoglobin (HbNO) have been investigated in the absence of any allosteric effector at pH 6.5 (in 0.1 M 2-[N-morpholino]ethanesulphonic acid/NaOH chloride-free buffer system), as well as at 100 K and/or 20 degrees C. Man and horse HbNO show spectroscopic properties that are generally taken as typical of the high affinity state of ferrous tetrameric Hb's; on the other hand, the spectroscopic properties of ruminant (i.e., buffalo, deer, mouflon, musk ox, ox, and reindeer) HbNO are characteristic of the low affinity conformation. These results are in keeping with the functional properties of the mammalian Hb's considered and have been related to the peculiar low oxygen affinity of ruminant Hb's.     

Ligand binding and hexacoordination in synechocystis hemoglobin.

A large and phylogenetically diverse group of organisms contain truncated hemoglobins, including the unicellular cyanobacterium Synechocystis (Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L., and Bolognesi, M. (2000) EMBO J. 19, 2424-2434). Synechocystis hemoglobin is also hexacoordinate, with a heme pocket histidine that reversibly coordinates the ligand binding site. Hexacoordinate hemoglobins are ubiquitous in plants and are now being identified in a diverse array of organisms including humans (Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant Physiol. 118, 1121-1125; Trent, J. T., III, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110). Rate constants for association and dissociation of the hexacoordinating amino acid side chain in Synechocystis hemoglobin have been measured along with bimolecular rate constants for association of oxygen and carbon monoxide following laser flash photolysis. These values were compared with ligand binding initiated by rapid mixing. Site-directed mutagenesis was used to determine the roles of several heme pocket amino acids in facilitating hexacoordination and stabilizing bound oxygen. It is demonstrated that Synechocystis hemoglobin contains a very reactive binding site and that ligand migration through the protein is rapid. Rate constants for hexacoordination by His(46) are also large and facilitated by other heme pocket amino acids including Gln(43).     

Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.