The effects of solvent on dioxygen binding to cobalt(II) picket fence porphyrins

The O₂ affinities on base adducts of four atropisomers of picket fence porphyrin Co(TpivPP), and the corresponding α⁴ complex containing valeramido pickets instead of pivalamido Co(α⁴-TvalPP) were measured in several solvents at O or −15 °C. The O₂ affinities of the α⁴ complexes are the lowest in DMF which is the most polar of the solvents used, while those of the other isomers are the highest in DMF. This observation was explained in terms of direct and indirect interactions between the solvent and the bound O₂. The trans-α² complex shows higher O₂ affinity in dichloromethane than those in aromatic solvents because of the preferential solvation of the deoxy complex in the solvents. The variation of the O₂ affinities of this system to solvents is considerably smaller than those of ‘flat porphyrin’ complexes. This result suggested that the pocket polarity introduced by the amide groups weakens the solvent–solute interaction on the O₂ affinities of this system and also that the solvation of the oxy state rather than the deoxy state predominantly affects the O₂ affinities. It was concluded that the enhanced O₂ uptake by the picket fence may be due to the stabilization of the oxy state by intramolecular interactions rather than to destabilization of the deoxy state by inhibiting solvation for the active site.     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. Reactions of peroxidase and myoglobin with oxygen, carbon monoxide and alkylisocyanides

The oxygen affinity of myoglobins increased in the order diacetyl- < chlorocruoro- < proto- < meso-myoglobins, which was a decreasing order of electron-withdrawing capacities of 2,4-substituents of deuteroheme. The results led to a conclusion that the π bonding predominates over the σ bonding in the myoglobin-oxygen interaction.On the contrary, the affinities of myoglobins and reduced peroxidases for ethylisocyanide decreased as the electron-withdrawing capacities of the substituents were decreased and it was therefore concluded that the σ bonding plays a dominant role in the ethylisocyanide binding to these hemoproteins.No simple relationship was observed in the cases of reduced peroxidase-CO complexes and myoglobin-isopropylisocyanide and -tertiarybutylisocyanide complexes. Among the hemoproteins tested, protohemoproteins appeared to have the lowest affinities for these ligands.As was the case with ligands such as azide and cyanide, the affinities of a deuterohemo-protein for oxygen, CO, and isocyanides were invariably much higher than the value expected from the Hammett-type relationship. The linear relationship between enthalpy and entropy was found to be applied for the reactions of ligands and hemoproteins including deutero derivatives and the irregularly high affinities of deuterohemoproteins for ligands could not be explained on the basis of thermodynamic analyses.     

Oxygenation of cobalt(II) protoporphyrin IX dimethyl ester bound to copolymer of 4-vinylpyridine and styrene

The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.     

The ground-state Na+ affinities of a series of substituted acetophenones: a DFT study

A detailed study of Na⁺ affinities of a series of para-substituted acetophenones and their O–Na⁺ counterparts was performed using density functional theory [Becke, Lee, Yang and Parr (B3LYP)] method using 6-311G(d,p) basis sets with complete geometry optimisation. The gas-phase O–Na⁺ complex formation turns out to be an exothermic case and the local stereochemical disposition of Na⁺ is found to be almost the same in each case. The presence of the para-substituent is seen to cause very little change in the Na⁺ affinity relative to the unsubstituted acetophenones. Electron-releasing p-substituents increase it by 0.0105 hartree and electron-withdrawing p-substituents decrease it by 0.011 hartree. Computed Na⁺ affinities are sought to be correlated with a number of computed system parameters such as the net charge on the Na⁺ and the carbonyl oxygen of the Na⁺ complexes and the net charge on the carbonyl oxygen of the free bases. The energetics, structural and electronic properties of the complexes indicate that the interaction between the Na⁺ ion and a carbonyl base is predominantly an ion–dipole attraction and the ion-induced dipole interaction as well rather than a covalent interaction.     

Thermodynamic resolution: How do errors in modeled protein structures affect binding affinity predictions?

The present study addresses the effect of structural distortion, caused by protein modeling errors, on calculated binding affinities toward small molecules. The binding affinities to a total of 300 distorted structures based on five different protein–ligand complexes were evaluated to establish a broadly applicable relationship between errors in protein structure and errors in calculated binding affinities. Relatively accurate protein models (less than 2 Å RMSD within the binding site) demonstrate a 14.78 (±7.5)% deviation in binding affinity from that calculated by using the corresponding crystal structure. For structures of 2–3 Å, 3–4 Å, and >4 Å RMSD within the binding site, the error in calculated binding affinity increases to 20.8 (±5.98), 22.79 (±11.3), and 29.43 (±11.47)%, respectively. The results described here may be used in combination with other tools to evaluate the utility of modeled protein structures for drug development or other ligand‐binding studies. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Decrease in oxygen affinity of myoglobin by formylation of vinyl groups of heme.

Three kinds of green synthetic myoglobin were prepared by recombination of horse heart apomyoglobin with spirographis (2-formyl-4-vinyl-), isospirographis (2-vinyl-4-formyl-), and 2,4-diformyldeuterohemins. The optical and oxygen binding properties of the reconstituted myoglobins containing two isomeric monoformyl-monovinylhemins were found to be different. The oxygen affinities (P50) of spirographis and 2,4-diformylmyoglobins are 2.7 and 2.8 mm Hg, respectively, at 25 degrees, and about 2.5 times lower than that of native protomyglobin, while that of isospirographis myoglobin is 1.0 mm Hg and is similar to native myoglobin. Spirographis oxymyoglobin has absorption maxima (alpha, beta, and Soret bands) at 601, 556.5, and 435 nm, isospirographis oxymyoglobin at 595, 550, 429 nm, and 2,4-diformyl oxymyoglobin at 603, 563.5, and 447 nm. The optical red shifts as well as the decrease in the oxygen affinities of these myoglobins are attributed mainly to the presence of strongly electron-attractive formyl side chains. Since the free isomers of monoformyl-monovinyl heme have similar properties, the differences observed after recombination with apoprotein must be caused by interactions with apomyoglobins. The degree of such a protein effect may be estimated by comparing the absorption spectra of heme before and after recombination and was found to differ among the various myoglobins. Comparison of the oxygen affinities of the myoglobins taking account of this protein factor showed that the increase in the P50 values are inversely related to that in the pK3 values of the free porphyrins. These results suggest the involvement of pi bonding in determining the oxygen-iron bond strength.     

Ligand-apomyoglobin interactions. Configurational adaptability of the haem-binding site.

1. The interaction of the haem-binding region of apomyoglobin with different ligands was examined by ultrafiltration, equilibrium dialysis and spectrophotometry, to study unspecific features of protein-ligand interactions such as they occur in, for example, serum albumin binding. 2. Apomyoglobin, in contrast with metmyoglobin, binds at pH 7, with a high affinity, one molecule of Bromophenol Blue, bilirubin and protoporphyrin IX, two molecules of n-dodecanoate and n-decyl sulphate and four molecules of n-dodecyl sulphate and n-tetradecyl sulphate. 3. The number of high-affinity sites and/or association constants for the alkyl sulphates are enhanced by an increase of hydrocarbon length, indicating hydrophobic interactions with the protein. 4. Measurements of the temperature-dependence of the association constants of the high-affinity sites imply that the binding processes are largely entropy-driven. 5. Binding studies in the presence of two ligands show that bilirubin plus Bromophenol Blue and dodecanoate plus Bromophenol Blue can be simultaneously bound by apomyoglobin, but with decreased affinities. By contrast, the apomyoglobin-protoporphyrin IX complex does not react with Bromophenol Blue. 6. Optical-rotatory-dispersion measurements show that the laevorotation of apomyoglobin is increased towards that of metmyglobin in the presence of haemin and protoporphyrin IX. Small changes in the optical-rotatory-dispersion spectrum of apomyoglobin are observed in the presence of the other ligands. 7. It is concluded that the binding sites on apomyoglobin probably do not pre-exist but appear to be moulded from predominantly non-polar amino acid residues by reaction with hydrophobic ligands. 8. Comparison with data in the literature indicates that apomyoglobin on a weight basis has a larger hydrophobic area avaialble for binding of ligands than has human serum albumin. On the other hand, the association constants of serum for the ligands used in this study are generally somewhat larger than those of apomyoglobin.     

Influence of steric factors on oxygen binding. I. Studies on 2,4-diisopropyldeuteroheme-myoglobin.

Sperm whale apomyoglobin was recombined with 2,4-diisopropyldeuterohemin to form 2,4-diisopropyldeuteroheme-myoglobin and its various physico-chemical properties were investigated to get an insight into the structural and functional role of the peripheral vinyl groups. 2,4-Diisopropyldeuteroheme-myoglobin showed a four times lower oxygen affinity at 25 degrees C and larger enthalpy and entropy changes of oxygenation than the corresponding values of native myoglobin. 2,4-Diisopropyldeuteroheme-metmyoglobin shows a pKa value of 9.68 which is higher than those of native metmyoglobin and mesoheme-metmyoglobin. The rate of autooxidation of oxy-form was about seven times larger in 2,4-diisopropyldeuteroheme-myoglobin than in native myoglobin. The electron-donating effect of isopropyl groups does not give straightforward explanation for these anomalous properties of 2,4-diisopropyldeuteroheme-myoglobin. It is proposed that site and stereospecific van der Waals' interaction between the polypeptide side chains and the peripheral 2,4-diisopropyl groups may weaken the interaction between the bound oxygen molecule and the distal His, resulting in the decrease in the stability of oxyform.     

Flexible docking using tabu search and an empirical estimate of binding affinity

This article describes the implementation of a new docking approach. The method uses a Tabu search methodology to dock flexibly ligand molecules into rigid receptor structures. It uses an empirical objective function with a small number of physically based terms derived from fitting experimental binding affinities for crystallographic complexes. This means that docking energies produced by the searching algorithm provide direct estimates of the binding affinities of the ligands. The method has been tested on 50 ligand-receptor complexes for which the experimental binding affinity and binding geometry are known. All water molecules are removed from the structures and ligand molecules are minimized in vacuo before docking. The lowest energy geometry produced by the docking protocol is within 1.5 Å root-mean square of the experimental binding mode for 86% of the complexes. The lowest energies produced by the docking are in fair agreement with the known free energies of binding for the ligands. Proteins 33:367–382, 1998. © 1998 Wiley-Liss, Inc.     

Adsorption of molecular oxygen by polymer covalently bonded co(II) porhyrin complex in toluene

Reaction betwenn molecular oxygen and polystyrene covalently bonded Co(II) protoporphyrin IX complex, which was prepaired by the incorporation of a cobaltous ion into the metal-free porphyrin polymer, was studied in the presence of N-ethylimidazole by measuring visible absorption and electron spin resonance spectra. It was found that the complex forms a monomeric oxygen adduct reversibly at low temperature dependent on oxygen pressure. In the presence of molecular oxygen, a new electron spin resonance signal due to the oxygen complex at g_iso=2.02 shows no superhyperfine splitting structure in fluid toluene solution even at ?80 °C, but it was observed in frozen toulene glass solution at ?120°C, The oxygen adducts of the complexes between C0(II) protoporphyrin IX dimethyl ester and N-ethylimidazole and copoly(styrene-N-vinylimidazole) showed eight resolved superhyperfine splitting at ?40 and ?60°C, respectively. The polymer covalently bonded Co(II) complex with N-ethylimidazole was oxidized at room temperature under oxygen atmosphere. It was suggested that a Co(II) porphyrin–oxygen adduct with an axial ligand may be oxidised monomolecularly at high temperature.     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. II. Reactions of peroxidases and metmyoglobin with azide, cyanide and fluoride

Artificial horseradish peroxidases and metmyoglobins were reconstituted from their apoproteins and the following unnatural hemes: mesoheme, deuteroheme, hematoheme, chlorocruoroheme, and diacetyldeuteroheme. The electron-withdrawing effects of the 2,4-substituents upon affinities of these hemoproteins for azide, cyanide, and fluoride were investigated. Peroxidase preparations used were acidic (A₁ + A₂) and neutral (B + C) enzymes.The electron withdrawal of the substituents increased the strength of ligaud binding. Plotting the logaritms of dissociation constants for the hemoprotein-ligand complexes against pK₃, a measure of relative basicities for metal-free porphyrins, the Hammett relationship was found to be applicable to this case except for a few. The affinities of deuterohemoproteins for ligands were definitely higher than those expected from the above relationship.The constant ρ, slope of the Hammett equation, varied with the ligand. For any particular ligand, the two peroxidase preparations gave the same ρ value, but it was different from the value for metmyoglobin.     

Feature selection and classification of protein–protein complexes based on their binding affinities using machine learning approaches

Protein–protein interactions are intrinsic to virtually every cellular process. Predicting the binding affinity of protein–protein complexes is one of the challenging problems in computational and molecular biology. In this work, we related sequence features of protein–protein complexes with their binding affinities using machine learning approaches. We set up a database of 185 protein–protein complexes for which the interacting pairs are heterodimers and their experimental binding affinities are available. On the other hand, we have developed a set of 610 features from the sequences of protein complexes and utilized Ranker search method, which is the combination of Attribute evaluator and Ranker method for selecting specific features. We have analyzed several machine learning algorithms to discriminate protein‐protein complexes into high and low affinity groups based on their K_d values. Our results showed a 10‐fold cross‐validation accuracy of 76.1% with the combination of nine features using support vector machines. Further, we observed accuracy of 83.3% on an independent test set of 30 complexes. We suggest that our method would serve as an effective tool for identifying the interacting partners in protein–protein interaction networks and human–pathogen interactions based on the strength of interactions. Proteins 2014; 82:2088–2096. © 2014 Wiley Periodicals, Inc.     

Binding, electrochemical activation, and cleavage of DNA by cobalt(II) tetrakis-N-methylpyridyl porphyrin and its beta-pyrrole brominated derivative

The binding of nucleic acids by water-soluble cobalt(II) tetrakis-N-methylpyridyl porphyrin, (TMPyP)Co, and its highly electron-deficient derivative cobalt(II) tetrakis-N-methyl pyridyl-beta-octabromoporphyrin, (Br(8)TMPyP)Co, was investigated by UV-visible absorption, circular dichroism (CD), and electrochemical and gel electrophoresis methods. The changes of the absorption spectra during the titration of these complexes with polynucleotides revealed a shift in the absorption maxima and a hypochromicity of the porphyrin Soret bands. The intrinsic binding constants were found to be in the range of 10(5)-10(6) M(-1). These values were higher for the more electron-deficient (Br(8)TMPyP)Co. Induced CD bands were noticed in the Soret region of the complexes due to the interaction of these complexes with different polynucleotides, and an analysis of the CD spectra supported a mainly external mode of binding. Electrochemical studies revealed the cleavage of polynucleotides by (TMPyP)Co and (Br(8)TMPyP)Co in the presence of oxygen preferentially at the A-T base pair region. Gel electrophoresis experiments further supported the cleavage of nucleic acids. The results indicate that the beta-pyrrole brominated porphyrin, (Br(8)TMPyP)Co, binds strongly and cleaves nucleic acids efficiently as compared with (TMPyP)Co. This electrolytic procedure offers a unique tool in biotechnology for cleaving double-stranded DNA with specificity at the A-T regions.     

Spectral and carbon monoxide binding properties of Fe(II) picket-fence porphyrin and protoheme bound to liposomes

Spectral and CO binding properties of liposome bound heme compounds, Fe(II) picket-fence porphyrin and protoheme, were examined. Phosphatidylethanolamine and phosphatidylcholine were used to make liposomes. Liposome-bound protoheme showed a very low CO affinity, whereas liposome-bound Fe(II) picket-fence porphyrin showed a high affinity. Addition of the ligand, 1-methylimidazole, modulated the CO affinities of both types of complexes to a level comparable to that of hemoglobin. However, autoxidation rates of the liposome bound heme compounds were still considerably high, and no stable oxygenated form could be observed.     

Relative affinity of 5,10-methylenetetrahydrofolylpolyglutamates for the Lactobacillus casei thymidylate synthetase-5-fluorodeoxyuridylate binary complex

The affinity of a series of methylenetetrahydrofolate polyglutamates for the binary complex of Lactobacillus casei thymidylate synthetase and fluorodeoxyuridylate has been investigated by kinetic and equilibrium techniques. The relative rates of binding for polyglutamates with one through seven glutamate residues were determined at 0 °C. Reactions were stopped by quenching into sodium dodecyl sulfate with subsequent determination of bound tritiated fluorodeoxyuridylate by gel filtration chromatography. Rates increased up to five residues beyond which a slight decrease occurred. Relative equilibrium binding affinities for all possible pairs of polyglutamates from the same series were determined by electrophoretic separation of tritiated complexes. In every case, the longer chain length member of the pair was bound more tightly. Complexes involving only a single subunit of thymidylate synthetase were compared with those in which both subunits were bound. Monomeric (1:1:1) complexes invariably showed a greater affinity for the longer chain length member of the pair than the corresponding dimeric (2:2:1) species. These results are interpreted in terms of possible binding site models for thymidylate synthetase.     

Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein–ligand interactions based on a study on caspase 3 inhibitors

Abstract

The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein–ligand complexes. CIFAP-2 method is established based on a protein–ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein–ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ?nference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein–ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3–ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC₅₀) of the CASP3 inhibitors.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Biochemical properties of the heme oxygenase inhibitor, Sn-protoporphyrin. Interactions with apomyoglobin and human serum albumin

Sn-protoporphyrin is a strong competitive inhibitor of heme oxygenase and a potential pharmacological agent for the treatment of neonatal hyperbilirubinemia. Little is otherwise known about the biochemistry of tin porphyrins. We have investigated aspects of the chemistry of tin-protoporphyrin in aqueous solution and of its interactions with heme-binding proteins other than heme oxygenase, specifically apomyoglobin and human serum albumin. In the pH region 7-10, Soret region absorption studies of unbound Sn-protoporphyrin demonstrate a pH-dependent monomer-dimer equilibrium (KD congruent to 10(6) M-1 at pH 7) with little higher aggregation. Dissociation of the dimer is relatively slow at neutral pH, permitting interaction of protein ligands with monomeric and dimeric species to be distinguished and providing insights into kinetic mechanisms of porphyrin binding by heme-binding proteins. In the present study, the kinetics of interaction of Sn-protoporphyrin with apomyoglobin are presented as novel evidence that this binding proceeds by an induced fit mechanism. Binding of Sn-protoporphyrin to both apomyoglobin and serum albumin is unexpectedly weak. Between pH 7 and 9, the apparent affinity of Sn-protoporphyrin for apomyoglobin is less than 1/200 that of heme and, at pH 9, is also significantly less than that of protoporphyrin. The apparent affinity of Sn-protoporphyrin for human serum albumin is less than 1/1000 that of heme and 1/30 to 1/100 that of protoporphyrin. Competition studies between heme and Sn-protoporphyrin and between bilirubin and Sn-protoporphyrin indicate that Sn-protoporphyrin distributes differently among porphyrin-binding sites on serum albumin than does heme and that it is also not an effective competitor with bilirubin for bilirubin-binding sites. These results argue that Sn-protoporphyrin should not significantly alter normal mechanisms for the binding and transport of heme or of preformed bilirubin by serum albumin. From a more general perspective, the results indicate potentially unusual binding site selectivity by tin chelates; possible origins of this selectivity are discussed.     

Syntheses and characterization of cobalt(II) complexes of dimeric ‘picket fence’ porphyrins

A series of dimeric picket fence porphyrinatocobalt(II) complexeses in which the length of the bridging chain controls the dioxygen affinity was newly derived from the coupling of two meso-mono- (β-o-aminophenyl)-tris-(α,α,α-o-pivaloylamidophenyl)- porphyrins with (CH₂)_n(COCl)₂ (n = 1, 3, 5 or 7). Some of the dimeric complexes form a unique ‘sandwich structure’ upon binding with certain bidentate ligands, and their dioxygen affinities are greatly increased compared with those for corresponding monomeric complexes. The relationship observed between the length of the bridging chain and the dioxygen affinity of the dimer complex having a sandwich structure is interpreted in terms of the displacement mechanism of the metal atom from a porphyrin plane.     

Oxygenation characteristics of the polymorphic hemoglobins of coho salmon (Oncorhynchus kisutch) at different developmental stages

• 1.1. The oxygen equilibria of the multiple hemoglobin of the fry and adult life stages of coho salmon (Oncorhynchus kisutch) were investigated. Components A 6–8 comprise 50–55% and >95% of the total hemoglobins of coho adults and fry respectively.
• 2.2. Fry hemoglobins exhibited a high oxygen affinity (P₅₀ = 3.9 mm Hg at 9.8°C, pH 8.5), a very large Bohr shift (ø = Δlog ₅₀/ΔpH = −1.729 at pH 7.1–7.5) which was non-linear in the pH range 6.8–8.5 and a large heat of oxygenation (ΔH = −20.8 kcal/mol).
• 3.3. Adult hemoglobins, however, exhibited a moderate oxygen affinity (P₅₀ = 14.0 mm Hg at 9.8°C, pH 8.2), a very small linear Bohr shift (ø = −0.172 at pH 7.0 −8.2) and a low heat of oxygenation (ΔH = −5.6- −7.5kcal/mol). Adenosine triphosphate had only minor influence on the oxygenation characteristics of adult heolyzates.
• 4.4. The results are discussed in terms of functional adaptation to environmental stresses and metabolic requirements.