The autoxidation and proton dissociation constants of tertiary diphosphines: relevance to biological activity

The pKas and autoxidation properties of a number of diphosphines which exhibit varying degrees of antitumor and cytotoxic activity were investigated. Titration by HClO4 in CH3NO2 was used to determine pKas of the following diphosphines: R2P(CH2)nPR'2, where for R = R' = Ph, n = 1, 2, and 3 (dppm, dppe, and dppp respectively); for R = R' = Et, n = 2 (depe); for R = Ph, R' = Et, n = 2 (eppe); and for cis and trans Ph2PCH = CHPPh2 (dppey). The difference between the first and second protonation constants decreases as the length of the carbon chain between the two phosphorus centers increases. Unsaturation in the carbon chain lowers pKas. -PEt2 centers are apparently more basic than -PPh2 centers. Apart from electrostatic effects, the protonation of a given phosphine center appears to be independent of the substituents at the second phosphine center. The autoxidation reactions of dppm, dppe, dppp, depe, and cis-dppey were studied in a variety of solvents by 31P NMR spectroscopy. The ethyl-substituted diphosphines were much more rapidly oxidized than the phenyl-substituted, and the pathways of autoxidation differed. Generally, the phenyl-substituted diphosphines gave only mono- and dioxides, while the ethyl-substituted diphosphines additionally gave phosphinites and other oxidation products. The relevance of the autoxidation reactivity and the pKas to the contrasting antitumor activity of these diphosphines is discussed.     

Synthesis and cytotoxicity of novel indirubin-5-carboxamides

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.     

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.     

Solution Structure and Acid-Base Properties of Reduced α-Conotoxin MI

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.     

P+QA- and P+QB- charge recombinations in Rhodopseudomonas viridis chromatophores and in reaction centers reconstituted in phosphatidylcholine liposomes. Existence of two conformational states of the reaction centers and effects of pH and o-phenanthroline

The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzyme inhibition by iminosugars: Analysis and insight into the glycosidase–iminosugar dependency of pH

Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme–inhibitor complexes with three (EH₃I), two (EH₂I), one (EHI), or no protons (EI), are possible. In the present work an analysis method is presented that from pH-inhibition data allows one to distinguish between the different complexes and determine which protonation state is preferred. It is also possible to determine the pH-independent binding constants of the inhibitor. Analysis of pH data for imino- and azasugar inhibition of β-glucosidases revealed that basic glycosidase inhibitors bind as the monoprotonated (EHI) complex. Three neutral inhibitors were also studied and two of these were also bound exclusively as the EHI complex while a third bound both as a EHI and a EH₂I complex.     

Nuclear-magnetic-resonance studies of 5'-ribonucleotide and 5'-deoxyribonucleotide conformations in solution using the lanthanide probe method.

The conformations of the metal-bound 5'-ribonucleotides and 5'-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Over-hauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5'-nucleotides, anti-(N in equalibrium S)-gg-g'g' and syn-(N in equalibrium S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.     

Charge recombination kinetics as a probe of protonation of the primary acceptor in photosynthetic reaction centers.

The kinetics of the charge recombination D+QA-----DQA was used to probe the protonation of the primary acceptor in reaction centers from Rhodopseudomonas sphaeroides, in which the native ubiquinone was replaced by anthraquinone. We found that QA- is stabilized by the rapid (t less than 10(-2) s) binding of a proton, with a pK of 9.8. The distance between QA- and the proton binding site was estimated to be larger than approximately 5 A.     

ATR-FTIR redox difference spectroscopy of Yarrowia lipolytica and bovine complex I

ATR-FTIR spectroscopy in combination with electrochemistry has been applied to the redox centers of Yarrowia lipolytica complex I. The redox spectra show broad similarities with previously published data on Escherichia coli complex I and with new data here on bovine complex I. The spectra are dominated by amide I/II protein backbone changes. Comparisons with redox IR spectra of small model ferredoxins demonstrate that these amide I/II changes arise primarily from characteristic structural changes local to the iron-sulfur centers, rather than from global structural alterations as has been suggested previously. Bands arising from the substrate ubiquinone were evident, as was a characteristic 1405 cm(-)(1) band of the reduced form of the FMN cofactor. Other signals are likely to arise from perturbations or protonation changes of a carboxylic amino acid, histidine, and possibly several other specific amino acids. Redox difference spectra of center N2, together with substrate ubiquinone, were isolated from those of the other iron-sulfur centers by selective redox potentiometry. Its redox-linked amide I/II changes were typical of those in other 4Fe-4S iron sulfur proteins. Contrary to published data on bacterial complex I, no center N2 redox-linked protonation changes of carboxylic amino acids or tyrosine were evident, and other residues that could provide its redox-linked protonation site are discussed. Features of the substrate ubiquinone associated with the center N2 spectrum were particularly clear, with firm assignments possible for bands from both oxidized and reduced forms. This is the first report of IR properties of ubiquinone in complex I, and the data could be used to estimate a stoichiometry of 0.2-0.4 per complex I.     

Proton-acceptor properties and capability for mutarotation of some glucosylamines in methanol

N-(m-Nitrophenyl)-beta-D-glucopyranosylamine (Gln), N-(N-methylphenyl)-beta-D-glucopyranosylamine (Glm), N-beta-D-glucopyranosylpyrazole (Glp), and N-beta-D-glucopyranosylimidazole (Gli) have been synthesized. Their basicity constants, pKb, determined in methanol were, respectively, 14.99, 14.36, 15.04, and 9.74. The derivatives of secondary amines (Glm, Glp, and Gli) did not mutarotate in methanol in the presence of 3,5-dinitrobenzoic acid and hydrochloric acid. The heats of formation and entropies were calculated by the AM1 and PM3 methods for the glucosylamines and their cations under consideration of two plausible protonation centers. Thermodynamic parameters for the proton transfer in the reaction: glucosylamine + CH3OH2+ = glucosylamineH+ + CH3OH were determined and the protonation center in the glucosylamine molecule was identified. The mechanism of mutarotation of the glucosylamines is discussed and the conclusion made that formation of an acyclic immonium cation is not a satisfactory condition for the reaction to proceed.     

Elementary reactions,structure-function relationships,and the potential relevance of low molecular weight metal-sulfur ligand complexes to biological N2 fixation

This article tries to rationalize the shortcomings of various model compounds and discusses requirements that a low-molecular compound must fulfill in order to become a potentially competitive catalyst for nitrogenases. For fundamental reasons, such a synthetic catalyst cannot be a precise structural duplicate of the active centers of nitrogenase. Results obtained with iron-sulfur carbonyl and diazene complexes further indicate that (1) the coupling and chronology of proton and electron transfer steps, (2) invariance of iron-sulfur distances within a wide range of electron density changes at the iron centers, and (3) Brönsted basic thiolate donors favoring the protonation of metal-sulfur cores and the formation of N–H···S bridges may be essential in order to reduce N₂ via N₂H₂ and N₂H₄ to NH₃ under mild conditions.     

Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones

Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications.     

Interplay between local protein interactions and water bridging of a proton antenna carboxylate cluster

Proteins that bind protons at cell membrane interfaces often expose to the bulk clusters of carboxylate and histidine sidechains that capture protons transiently and, in proton transporters, deliver protons to an internal site. The protonation-coupled dynamics of bulk-exposed carboxylate clusters, also known as proton antennas, is poorly described. An essential open question is how water-mediated bridges between sidechains of the cluster respond to protonation change and facilitate transient proton storage. To address this question, here I studied the protonation-coupled dynamics at the proton-binding antenna of PsbO, a small extrinsinc subunit of the photosystem II complex, with atomistic molecular dynamics simulations and systematic graph-based analyses of dynamic protein and protein-water hydrogen-bond networks. The protonation of specific carboxylate groups is found to impact the dynamics of their local protein-water hydrogen-bond clusters. Regardless of the protonation state considered for PsbO, carboxylate pairs that can sample direct hydrogen bonding, or bridge via short hydrogen-bonded water chains, anchor to nearby basic or polar protein sidechains. As a result, carboxylic sidechains of the hypothesized antenna cluster are part of dynamic hydrogen bond networks that may rearrange rapidly when the protonation changes.     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.     

Scaled quantum mechanical force field for alanyl-alanine peptide in solution

We have obtained ab initio scale factors and assigned frequencies for the alanine-alanine peptide in water. Calculations were performed on the isolated acidic and basic Ala-Ala structures, two one-water basic Ala-Ala supermolecules, and one two-water acidic and one two-water basic Ala-Ala supermolecules. Force constants were scaled using the experimentally determined Raman and Fourier transform infrared vibrational frequencies of four isotopic species of Ala-Ala in water at pH 13 and pH 1. Most of the 4-31G scale factors were transferable from smaller molecules. All but one scale factor were directly transferable between the pH 1 and pH 13 species for coordinates unchanged by protonation in both the isolated and two-water supermolecule structures. Scale factors for nonpolar coordinates were transferable between all Ala-Ala species with only a few small changes. Good agreement was obtained between the calculated and experimental frequencies for all isotopic species and structures.     

Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion

We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.     

Acid-base properties of ionophore A23187 in methanol-water solutions and bound to unilamellar vesicles of dimyristoylphosphatidylcholine

The acid-base properties of ionophore A23187 in methanol-water solutions (0--95% w/w) and bound to unilamellar vesicles of dimyristoylphosphatidylcholine were examined by ultraviolet and fluorescence spectroscopy, and the spectral properties for the acidic and basic forms were defined under these conditions. Standard mixed-solvent buffers were employed to calibrate pH measurement in the methanol-water solvents. In 65% methanol-water, two protonation equilibria were observed, the most basic of which displayed a value for the logarithm of the protonation constant (log KH) of 7.19 +/- 0.05 at 25 degrees C and 0.05 M ionic strength. Instability of A23187 was encountered below pH approximately 4; however, decomposition was slow enough to allow log KH for the more acidic equilibrium to be estimated as 1.28. Comparison of these results to those obtained with the methyl ester of A23187 (log KH = 1.32) and literature values for other model compounds allowed assignment of the more basic equilibrium to the carboxylic acid moiety and the more acidic one to the N-methylamino substituent of the benzoxazole ring. log KH of the carboxylic acid increased from 5.69 +/- 0.05 to 9.37 +/- 0.05 over the range of solvent polarity encompassed by water to 95% methanol-water. Values for the ground state (absorption) and first excited state (fluorescence) were equal within experimental error. The logarithm of the protonation constant for the membrane-bound ionophore, measured under conditions where the surface potential generated by ionization did not significantly alter the equilibrium, was found to be 7.85 +/- 0.05 at 25 degrees C and at ionic strength of 0.05 M in the aqueous phase. The value agrees with that observed in 80% methanol-water, as does the wavelength of maximum fluorescence emission for the membrane-bound free acid. An interfacial location for the monoprotonated form of the benzoxazolate moiety is proposed, both above and below the membrane phase transition temperature. The location of other regions of the A23187 molecule could not be assessed from these data.     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.     

Gas-phase acidity and dynamics of the protonation processes of carbidopa and levodopa. A QM/QD study

The present work details, our efforts to investigate and compare the acid–base properties of levodopa (LD) and carbidopa (CD), a drug combination being used in the treatment of Parkinson’s disease. Protonation and deprotonation were examined for all possible sites in the two drugs. Proton affinity and proton detachment enthalpies were computed at the B3LYP/6–311++G** level of theory. Results of the present work reveal that CD is more basic and can abstract protons in solution much more efficiently than LD. Furthermore, at all deportation sites considered, CD is more acidic than LD. DFT-based ADMP, dynamic simulations have been performed to explore the dynamics of the protonation processes in LD and CD. Thus, while the dynamics of the protonation process of LD is very straightforward leading to the formation of the corresponding cation, the protonation process in CD is very complex involving a major geometry change and rearrangement. Results of the present work reveal that the active species in acid medium is not CD in its normal geometry but a carbonyl hydrazine form instead. The presence of the carbonyl group β to the hydrazine group may very well underlie its enhanced activity which allows it to bind to the active site of the DDC enzyme. The relative stabilities of various water–water–CD complexes have been computed and compared.