X-ray investigation of the binding of 1,10-phenanthroline and imidazole to horse-liver alcohol dehydrogenase

We have studied the binding of two inhibitor molecules, imidazole and 1,10-phenanthroline, to liver alcohol dehydrogenase by crystallographic methods. X-ray data for the imidazole complex were collected to 0.29-nm resolution and for the 1,10-phenanthroline complex to 0.45-nm resolution. In both cases we found only one peak in the difference electron density maps close to the active zinc atom. The peak corresponding to 1,10-phenanthroline overlaps the site of the density of the zinc-bound water in the apoenzyme and the imidazole density partly overlaps this density. We can not discern any additional peaks close to the zinc atom which would correspond to new positions of bound water. We thus conclude that both these inhibitors bind to the catalytic zinc atom and that upon binding they displace the water molecule that is firmly bound to this zinc atom in the apoenzyme. We do not see any structural changes in the remaining part of the molecule.     

The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

Circular dichroic properties and conformation of thionicotinamide dinucleotides bound to horse-liver alcohol dehydrogenase.

The interaction between horse liver alcohol dehydrogenase and the oxidized and reduced forms of the 3-thionicotinamide--adenine dinucleotide coenzyme analogues (sNAD and sNADH) has been investigated by ultraviolet absorption, fluorescence and circular dichroism. The fluorescence of sNADH is enhanced when bound to the enzyme, and the protein fluorescence is quenched by both sNADH (60--65%) and sNAD (65%). The possible origin of the larger quenching produced by sNAD with respect to that of NAD is discussed. Coenzyme dissociation constants have been determined by monitoring the quenching of the protein fluorescence, and some kinetic consequences of these dissociation constants are discussed. The dichroic properties of various enzyme complexes have been investigated, and are discussed in terms of conformational changes and environmental changes during coenzyme binding. The conformation of sNAD bound to the enzyme in the presence of trifluorethanol and the conformation of sNADH bound to the enzyme in the presence of isobutyramide have been analyzed in particular detail. Also the circular dichroic spectrum of the apoenzyme is discussed in terms of contributions of the aromatic amino acid residues in the highly resolved 240--310-nm region and in terms of helix content in the 220-nm region.     

DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline

Absorption spectroscopy and circular dichroism (CD) have been used to characterize the DNA binding of [Fe(phen)3]2+, [Fe(phen)2(DIP)]2+ and [Fe(phen)(DIP)2]2+ where phen and DIP stand for 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline, respectively. Both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ bind weakly to calf thymus DNA (CT-DNA) in an electrostatic mode, while [Fe(phen)(DIP)2]2+ binds more strongly to CT-DNA, possibly in an intercalation mode. The hypochromicity, red shift and Kb increase in the order [Fe(phen)3]2+ < [Fe(phen)2(DIP)]2+ < [Fe(phen)(DIP)2]2+ in accordance with the increase in size and hydrophobicity of the iron(II) complexes. The thermodynamic parameters obtained suggest that the DNA binding of both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ is entropically driven, while that of [Fe(phen)(DIP)2]2+ is enthalpically driven. A strong CD spectrum in the UV and visible region develops upon addition of CT-DNA into the racemate solution of each iron(II) complex (Pfeiffer effect). This has revealed that a shift in diastereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA-complex diastereomers. The striking resemblance of the CD spectral profiles to those of the pure delta-enantiomer indicates that the delta-enantiomer of the iron(II) complexes is preferentially bound to CT-DNA. The mechanism of the development of Pfeiffer CD is proposed on the basis of kinetic studies on the DNA binding of the racemic iron(II) complexes.     

The reaction of horse-liver alcohol dehydrogenase with glyoxal.

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.     

A new polymorph of dichlorido(1,10-phenanthroline)platinum(II)

A yet unreported polymorph of [PtCl₂(1,10-phenanthroline)] was obtained by slow decomposition, in CH₂Cl₂ solution, of [Pt{CH₂CH₂N(CH₂CH₃)₂-κC,κN}(1,10-phenanthroline)](ClO₄). The structure of the new orthorhombic form, III, (space group Pna2₁), is described and compared to those of the two already reported forms, I and II, which are monoclinic (space group P2₁/c) and orthorhombic (space group Pca2₁), respectively [21]. Polymorph III appears to be the least stable of the three.     

Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species.

Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.     

Synthesis, characterization and biological properties of cobalt(II) complexes of 1,10-phenanthroline and maltol

The synthesis and characterization of two cobalt(II) complexes, Co(phen)(ma)Cl 1 and Co(ma)₂(phen) 2, (phen = 1,10-phenanthroline, ma⁻ = maltolate or 2-methyl-4-oxo-4H-pyran-3-olate) are reported herein. The complexes have been characterized by FTIR, CHN analysis, fluorescence spectroscopy, UV-visible spectroscopy, conductivity measurement and X-ray crystallography. The number of chelated maltolate ligands seems to influence their DNA recognition, topoisomerase I inhibition and antiproliferative properties.     

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when epsilon is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.     

Horse liver alcohol dehydrogenase derivatives containing nickel(II) and cobalt(lI) in the noncatalytic metal binding site

The noncatalytic zinc in horse liver alcohol dehydrogenase was selectively replaced by nickel(II). This novel species, Zn(c)₂Ni(n)₂⁴ horse liver alcohol dehydrogenase (where c denotes the catalytic and n denotes the noncatalytic site) was compared to Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase with respect to its absorption, circular dichroism and magnetic circular dichroism spectra, as well as its magnetic moment. For Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase (prepared according to refs. 1 and 2) the extinction coefficients were redetermined in the UV, visible and near-infrared region and the molar ellipticities in the range 300-800 nm. The average magnetic moment was determined by the NMR method as 4.5-5.0 B.M. The results confirm a tetrahedral structure in the zinc-cobalt enzyme. In contrast, the spectroscopic data and the zero magnetic moment support a planar geometry for the nickel(Il) bound in the noncatalytic site. Zn(c)₂Ni(n)₂ horse liver alcohol dehydrogenase is very temperature-sensitive and precipitates after short exposure to room temperature. Stored in the cold it has the same activity as the native enzyme. The results indicate that the protein is flexible in the loop region binding the noncatalytic metal ion and that it may retain catalytic activity even in a partially distorted conformation.     

DNA binding of iron(II) complexes with 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline: salt effect,ligand substituent effect,base pair specificity and binding strength

The DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline(phen) and 4,7-diphenyl-1,10-phenanthroline(dip), [Fe(phen)(3)](2+), [Fe(phen)(2)(dip)](2+) and [Fe(phen)(dip)(2)](2+) has been characterized by spectrophotometric titration and melting temperature measurements. The salt concentration dependence of the binding constant has allowed us to dissect the DNA-binding constant and free energy change of each iron(II) complex into the nonelectrostatic and polyelectrolyte contributions. A comparison of the nonelectrostatic components in the binding free energy changes among iron(II) complexes has made it possible to rigorously evaluate the contribution of the ligand substituents to the DNA-binding event. The peripheral substitution of phen by two phenyl groups increases the nonelectrostatic binding constant of the iron(II) complex more than 20 times, which is equivalent to approximately 7.5 kJ mol(-1) of more favorable contribution to the DNA binding. In general, the iron(II) complexes studied have higher affinity towards the more facile A-T sequence than the G-C sequence. This preferential binding may be attributed to the steric effect induced by the ancillary part of the ligands in the course of DNA binding. The binding of disubstituted iron(II) complex to DNA is quite strong as reflected in the modest increase in the denaturation temperature (T(m)) of double helical DNA upon the interaction with the iron(II) complex.     

Carboxymethylation of horse-liver alcohol dehydrogenase in the crystalline state. The active-site zinc region and general anion-binding site of the enzyme correlated in primary and teritiary structures.

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)2-/4 , 1,10-phenanthroline, Au(CN)-/2 and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboyxmethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN2-/4 to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN2-/4 and to a lesser extent Au(CN)-/2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identificationof this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.     

Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH

Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with lambda max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2'-trifluoroethoxide ion (TFE-), CoIIE(NAD+, TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+, RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+, RCH2O-) in equilibrium CoIIE(NADH, RCHO). From competition experiments, we infer that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+, RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+, RCH2OH) in equilibrium CoIIE(NAD+, RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of an intermediate. Via spectral comparisons with CoIIE-(NAD+, TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same CoIIE(NAD+, RCH2O-) ternary complex detected during alcohol oxidation.     

Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.     

Synthesis and DNA binding of mu-[2,9-bis(2-imidazo[4,5-f][1,10]phenanthroline)-1,10-phenanthroline]bis[1,10-phenanthrolinecopper(II)

A binuclear complex [(phen)Cu(mu-bipp)Cu(phen)](ClO(4))(4), where phen=1,10-phenanthroline and bipp=2,9-bis(2-imidazo[4,5-f][1,10]phenanthroline)-1,10-phenanthroline, has been synthesized and its interaction with calf-thymus DNA in the buffer containing 5mM Tris and 50mM NaCl has been studied by means of electronic absorption titration, luminescence titration and viscometric measurements. The absorbance of the complex in the range of 320-400 nm, which is mainly based on bipp showed no obvious change upon addition of DNA, while the peak at 270 nm, which is determined by both phen and bipp decreased by up to 18%. The emission band of the complex around 360 nm decreased remarkably in presence of DNA. The emission quenching of this complex by [Fe(CN)(6)](4-) was depressed greatly when bound to DNA. The relative viscosity of DNA was increased by this complex more significantly than a bipp directed intercalating reagent. These results suggest that this complex binds to calf thymus DNA by intercalation of the two phenanthrolinecopper terminals. The apparent intrinsic binding constant of the complexes with DNA was 1.6 x 10(4)M(-1) as determined by UV-visible titration.