Oxidation of D-glucose in the presence of 2,2'-bipyridine by CrVI in aqueous micellar media: a kinetic study

The kinetics of Cr(VI) oxidation of D-glucose to the corresponding lactone in the presence and absence of 2,2'-bipyridine (bipy) has been carried out under the conditions, [D-glucose](T) > [Cr(VI)](T) at different temperatures in aqueous micellar media. The monomeric Cr(VI) species has been found to be kinetically active in the absence of bipy whereas in the bipy-catalysed path, the Cr(VI)-bipy complex has been found to be the active oxidant. In the bipy-catalysed path, the Cr(VI)-bipy complex undergoes nucleophilic attack by the substrate to form a ternary complex. The ternary complex spontaneously experiences a redox decomposition (through two-electron transfer) in the rate-determining step leading to the product lactone and Cr(IV)-bipy complex. The Cr(IV)-bipy complex then takes part in faster steps in the further oxidation of D-glucose and is ultimately converted into a Cr(III)-bipy complex. In the uncatalysed path, the Cr(VI)-substrate ester experiences acid catalysed redox decomposition (two-electron transfer) in the rate-determining step. The uncatalysed path shows a second order dependence on [H(+)] and a first order dependence on each of the reactants [D-glucose](T) and [Cr(VI)](T). In contrast, the bipy-catalysed path shows a first order dependence on each of the reactants [H(+)], [D-glucose](T) and [Cr(VI)](T). The bipy-catalysed path is first order in [bipy](T). These observations remain unaltered in the presence of externally added surfactants. The effect of the cationic surfactant, N-cetylpyridinium chloride (CPC) and anionic surfactant, sodium dodecyl sulfate (SDS) on both the uncatalysed and bipy-catalysed path has been studied. CPC inhibits both the uncatalysed and bipy-catalysed path, while SDS catalyses these reactions. The observed micellar effects have been explained by considering hydrophobic and electrostatic interactions between the surfactants and reactants.     

The effects of ligand substitution and deuteriation on the spectroscopic and photophysical properties of [Ru(LL)(CN)4]2- complexes.

The spectroscopic characterisation of a series of [Ru(LL)(CN)(4)](2-) complexes, where LL = 1,10-phenanthroline (phen) and its methyl- and phenyl-substituted derivatives and several deuteriated isotopologues are reported. The optical and vibrational properties of these complexes are compared with that of the series of 2,2'-bipyridine (bipy) derivatives and analogous [Ru(LL)(3)](2+) complexes. It has been demonstrated that substitution at the 4,4' positions of bipy and 4,7-positions of phen by electron donating (CH(3)) and withdrawing (C(6)H(5), COO(-)) groups induces a pronounced blue and red shift, respectively, in the lowest energy (1)MLCT absorption band of [Ru(LL)(CN)(4)](2-). The energy of the emission originating from the (3)MLCT excited state is found to be dependant on the nature of the vibrational modes of the aromatic rings and the electron donating and/or withdrawing properties of the substituents. Single-mode Franck-Condon analysis indicates that methyl substitution leads to a significant increase in the Huang-Rhys factor (S(M)), while phenyl substitution results in a decrease in S(M) for both series (bipy and phen) of complexes. The rate of non-radiative (k(nr)) and radiative decay (k(ph)) to the ground state and the parameters of thermally activated deactivation pathways (A(4th), DeltaE(4th) and A(dd), DeltaE(dd)) were estimated from the temperature dependence of luminescence quantum yields and lifetimes. It has been demonstrated that the non-radiative decay rate and the temperature dependent decay processes are more efficient for bipy complexes than for phen derivatives due to the rigidity of the latter ligand.     

Syntheses, spectra and crystal structures of ruthenium(II) complexes with polypyridyl: [Ru(bipy)2(phen)](ClO4)2 · H2O and [Ru(bipy)2(Me-phen)](ClO4)2

Two ruthenium(II) complexes with polypyridyl, Ru(bipy)₂(phen)](ClO₄)₂·H₂O (1) and [Ru(bipy)₂(Me-phen)](ClO₄)₂ (2), (phen = 1,10-phenanthroline, bipy = 2,2′-bipyridine, Me-phen = 5-methyl-1,10-phenanthroline), were synthesized and characterized by IR, MS and NMR spectra. Their structures were determined by single crystal X-ray diffraction techniques. The strong steric interaction between the polypyridyl ligands was relieved neither by the elongation of the Ru---N bonds nor increase of the N---Ru---N bite angles. The coordination sphere was distorted to relieve the ligand interaction by forming specific angles (δ) between the polypyridyl ligand planes and coordination planes (N---Ru---N), and forming larger twisted angles between the two pyridine rings for each bipy. The bond distances of Ru---N(bipy) and Ru---N(phen) were virtually identical with experimental error, as expected of π back-bonding interactions which statistically involve each of the ligands present in the coordination sphere.     

Copper(II) pyridinecarboxylate adducts with chelating ligands as potential antimicrobial agents

The synthesis and characterization of seven new solid complexes, [Cu(2-MeSnic)₂ (phen)] (2-MeSnic = 2-methylthionicotinate, phen = 1,10-phenanthroline), [CuX₂(bipy)(H₂O)] (X = 2-MeSnic or nic (nicotinate), bipy = 2,2′-bipyridine), [Cu(isonic)₂(bipy)(H₂O)] · H₂O (isonic = isonicotinate), [Cu(bipy)₂(H₂O)](2-MeSnic)₂ · 3H₂O, [Cu(phen)₂(H₂O)](isonic) ₂ · 2H₂O and [Cu(phen)₂(H₂O)](nic)₂ · 3H₂O, are reported. The composition and stereochemistry as well as the mode of ligand coordination have been determined by elemental analysis, IR, electronic and EPR spectra. The carboxyl group of the pyridinecarboxylate anions coordinates to the Cu(II) atom as an unidentate or as a chelating ligand. The EPR spectra of studied complexes are monomeric except for the spectrum of [Cu(2-MeSnic)₂(bipy)(H₂O)], which shows triplet state feature. Half-field transition, observed for [Cu(2-MeSnic)₂(bipy)(H₂O)], [Cu(bipy)₂(H₂O)](2-MeSnic)₂ · 3H₂O and [Cu(phen)₂(H₂O)](nic)₂ · 3H₂O, was used to estimate the interspin copper-copper distances. In all cases, the available evidence supports square-pyramidal environment about the copper(II) atom, which is confirmed by crystal and molecular structure of one of the products, namely [Cu(2-MeSnic)₂(bipy)(H₂O)]. The antimicrobial effects have been tested on various strains of bacteria, yeasts and filamentous fungi.     

Fluorous interactions in complexes of lead(II) hexafluoroacetylacetonate

Structure determinations for 2,2′-bipyridine and 1,10-phenanthroline adducts of lead(II) hexafluoroacetylacetonate, [Pb(bipy)₂(hfacac)₂] (1), [Pb(bipy)(hfacac)₂] (2), and [Pb(phen)(hfacac)₂] (3), show that the balance of intermolecular forces within the lattices is seemingly sensitive to the adduct stoichiometry but not to the nature of the heteroaromatic base. In 3, a structure, in which there is an apparent preference for CF/aromatic interactions over separate CF/CF and aromatic/aromatic interactions, is essentially identical at both 120 and 293 K.     

Saccharinate-metal complexes with 1,10-phenanthroline (phen) or 2,2′-bipyridine (bipy) as co-ligands; the synthesis, crystal and molecular structures of five new compounds of divalent metals

Five new saccharinate complexes of divalent metals with either phen or bipy as co-ligands have been synthesised, and fully characterised by single crystal X-ray diffraction at low temperature. The complexes [M(phen)₂(H₂O)₂](sac)₂·H₂O (M = Co or Zn) are isostructural, while [Hg(bipy)₂(sac)₂] is isostructural with the analogous cadmium(II) compound, which has been described previously in the literature. Cadmium(II) complex [Cd(phen)₂(sac)(H₂O)](sac).H₂O has an octahedral cation with the unidentate ligands in cis-positions, while [Hg(phen)₂(sac)](sac)·2.5 H₂O provides a rare example of a distorted tbp structure for the cation, with five nitrogen donors. The structures are compared with those of related saccharinate complexes. In general, the more sterically demanding phen, when compared with bipy, forces more of the saccharinate anions to be uncoordinated, and for smaller ligands such as H₂O to be coordinated to the metal, despite the electrostatic attraction between the positively charged metal and the anion. Intramolecular hydrogen bonding involving saccharinate groups plays an important role in all the hydrated complexes.     

Picosecond Kerr-gated time-resolved resonance Raman spectroscopy of the [Ru(phen)(2)dppz](2+) interaction with DNA

To investigate the basis of the 'light-switch' effect, the solvent dependence of the Kerr-gated picosecond-time resolved resonance Raman (TR(3)) spectra of [Ru(bpy)(2)dppz](2+), [Ru(phen)(2)dppz](2+), and the modified complex [Ru(phen)(2)cpdppzOMe](2+) and a dimer [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) were studied. The investigation focussed on comparing the behaviour of [Ru(phen)(2)dppz](2+) in acetonitrile, ethanol, H(2)O, D(2)O, and DNA. The data are consistent with a model wherein excitation induces metal-to-ligand charge transfer (MLCT) to any of the ligands (termed the 'precursor' state) which, by interligand electron transfer (ILET), produces an excited state localised on the dppz ligand, MLCT(1). In water this state relaxes with a characteristic time of approximately 6 ps to a non-emissive state (MLCT(2)). The TR(3) spectra in water, acetonitrile and DNA are all distinctly different. However, the early (4 ps) water spectrum resembles the spectrum in DNA. This interesting observation suggests that the DNA-bound excited state of the complex can be thought of as a model for the initial, poorly solvated state in water.     

Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization

Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P](rm,max) was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P](rm). In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P](rm) --> [P](rm,max). In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P](rm). (c) 1995 John Wiley & Sons Inc.     

Magnetic, spectroscopic, structural and biological properties of mixed-ligand complexes of copper(II) with N,N,N,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands

Two new mixed ligand complexes of copper(II) with N,N,N^′,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands have been prepared and characterized by means of spectroscopic, magnetic and single-crystal X-ray diffraction methods. These two complexes are isomorph and isostructure in which the coordination polyhedron about the copper(II) ion is distorted square pyramidal. [Cu(PMDT)(bipy)]²⁺ and [Cu(PMDT)(phen)]²⁺ show an absorption wavelength maximum at 625 and 678 nm, respectively, assigned to the d-d transition. Antibacterial, antifungal and superoxide dismutase activities of these complexes have also been measured. It was observed that [Cu(PMDT)(bipy)](ClO₄)₂ was more effective against P. Pyocyanea and Klebsiella sp. than S. aureus. Similarly, Fusarium sp. was highly susceptible against [Cu(PMDT)(bipy)](ClO₄)₂ but less susceptible against [Cu(PMDT)(phen)](ClO₄)₂.     

Aerobic Cr(VI) reduction by Thermus scotoductus strain SA-01

AIM: To evaluate Thermus scotoductus SA-01's ability to reduce Cr(VI) aerobically. METHODS AND RESULTS: T. scotoductus SA-01 is able to reduce Cr(VI) aerobically when grown in a complex organic medium containing Cr(VI) concentrations up to 0.5 mmol l(-1). Suspension of T. scotoductus SA-01 cells also reduced Cr(VI) aerobically under nongrowth conditions using a variety of electron donors as well as in the absence of an exogenous electron donor. The optimum temperature and pH for Cr(VI) reduction under nongrowth conditions were found to be 80 degrees C and 7, respectively. It was also found that the Cr(VI) reduction was catalysed by a cytoplasmic, constitutively expressed enzyme. CONCLUSIONS: Apart from SA-01's ability to reduce Cr(VI) through a strictly anaerobic membrane-bound mechanism (unpublished data), it also has a second enzyme localized in the cytoplasm that can reduce Cr(VI) aerobically. As this enzyme is constitutively expressed and not induced by Cr(VI), it remains to be determined whether it has any other physiological functions. SIGNIFICANCE AND IMPACT OF THE Study: This is the first report of a Thermus species able to reduce Cr(VI) aerobically and extends the knowledge of parameters associated with Cr(VI) reduction. Employing thermophiles in bioremediation using industrial bioreactors would cancel the need for expensive cooling systems.     

Synthesis, DNA-binding and photocleavage studies of cobalt(III) mixed-polypyridyl complexes: [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+)

Two novel cobalt(III) mixed-polypyridyl complexes [Co(phen)(2)(dpta)](3+) and [Co(phen)(2)(amtp)](3+) (phen=1,10-phenanthroline, dpta=dipyrido-[3,2-a;2',3'-c]- thien-[3,4-c]azine, amtp=3-amino-1,2,4-triazino[5,6-f]1,10-phenanthroline) have been synthesized and characterized. The interaction of these complexes with calf thymus DNA was investigated by spectroscopic, cyclic voltammetry, and viscosity measurements. Results suggest that the two complexes bind to DNA via an intercalative mode. Moreover, these Co(III) complexes have been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365nm. The mechanism studies reveal that hydroxyl radical (OH()) is likely to be the reactive species responsible for the cleavage of plasmid DNA by [Co(phen)(2)(dpta)](3+) and superoxide anion radical (O(2)(-)) acts as the key role in the cleavage reaction of plasmid DNA by [Co(phen)(2)(amtp)](3+).     

A study of the interaction of Cr(III) complexes and their selective binding with B-DNA: a molecular modeling approach

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H(2)O)(2)](+); salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H(2)O)(2)](+); salprn denotes 1, 3 bis- salicylideneaminopropane, [Cr(phen)(3)](3+); phen denotes 1, 10 phenanthroline and [Cr(en)(3)](3+); en denotes ethylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)(12), d(CGCGAATTCGCG)(2) and d(GC)(12) sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

The light switch effect upon the association of [Ru(1,10-phenanthroline)2dipyrido[3,2-a:2',3'-c]phenazine]2+ with single stranded poly(dA) and poly(dT)

Large enhancement in the luminescence intensity of the Delta- and Lambda-Ru(phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) complexes upon their association with single stranded poly(dA) and poly(dT) is reported in this work. As the mixing ratio ([[Ru(phen)(2)DPPZ](2+)]/[DNA base]) increases, the luminescence intensity increase in a sigmoidal manner, indicating that the enhancement involves some cooperativity. At a high mixing ratio, the luminescence properties are affected by the nature of the DNA bases and not by the absolute configuration of the [Ru(phen)(2)DPPZ](2+) complex, indicating that the single stranded poly(dA) and poly(dT) do not recognize the configuration of the metal complex. In the case of the Lambda-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex, the manner of the enhancement is somewhat different from the other Ru(II) complex-polynucelotide combinations: the luminescence intensity reached a maximum at an intermediate mixing ratio of 0.32, and gradually decreased as the mixing ratio increased. In contrast to other complexes at high mixing ratios, an upward bending curve was found in the Stern-Volmer plot, which indicates that the micro-environment of the Lambda-[Ru(phen)(2)DPPZ](2+) is heterogeneous. In the Delta-[Ru(phen)(2)DPPZ](2+)-poly(dT) complex case, formation of this highly luminescent species at an intermediate mixing ratio is far less effective.     

Synchronous fluorescence, UV-visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.     

A bioprocessing strategy that allows for the selection of Cr(VI)-reducing bacteria from soils

Anaerobic bacteria that reduce hexavalent chromium [Cr(VI)] to trivalent [Cr(III)] are common in soils and were used to develop a bioprocess employing a selection strategy. Indigenous Cr(VI)-reducers were enriched from Cr(VI)-contaminated soil under anaerobic conditions. The mixed culture was then tested for Cr(VI)-reducing activity in a chemostat, followed by transfer to a 1-L packed-bed bioreactor operated at 30°C for additional study. The support material used in the reactor consisted of 6-mm porcelain saddles. Cr(VI) concentrations in the liquid ranged from 140–750 mg L⁻¹. Cr(VI)-reducing bacteria were the dominant population with Cr(VI)-reduction rates of approximately 0.71 mg g⁻¹ dry cells h⁻¹ achieved at Cr(VI) concentrations of 750 mg L⁻¹. These results indicate a potential for selecting and maintaining indigenous Cr(VI)-reducers in a bioreactor for Cr(VI)-remediation of groundwater or soil wash effluents. Received 09 January 1996/ Accepted in revised form 15 November 1996     

Syntheses and DNA-binding studies of two ruthenium(II) complexes containing one ancillary ligand of bpy or phen: [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)2

Two new ruthenium(II) complexes of [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)(2) (bpy=2,2'-bipyridine, phen=1,10-phenanthroline, pp[2,3]p=pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) have been synthesized and characterized by elemental analysis and 1H NMR spectra. The calf thymus DNA-binding properties of the two complexes were investigated by UV-visible and emission spectroscopy, competitive binding experiments with ethidium bromide and viscosity measurements. The results indicate that the two complexes intercalate between the base pairs of the DNA tightly with intrinsic DNA-binding constants of 3.08 x 10(6) and 6.53 x 10(6) M(-1) in buffered 50 mM NaCl, respectively, which are much larger than 6.9 x 10(5) M(-1) for [Ru(bpy)2(pp[2,3]p)](ClO4)2 containing two ancillary ligands of bpy.     

Kinetics and mechanism of oxidation of d-erythrose and dL-glyceraldehyde by chromium(VI) and vanadium(V) in perchloric acid medium

The kinetics of oxidation of d-erythrose and dL-glyceraldehyde by chromium (VI) and vanadium(V) in perchloric acid medium have been investigated spectrophotometrically. Each reaction was first-order with respect to [oxidant] and [substrate]. The reactions were catalysed by acid, but their dependence on acidity was complex. Sodium perchlorate accelerated the rate of each reaction. The oxidation rates follow the order glyceraldehyde > erythrose. The activation parameters were calculated and mechanisms consistent with the experimental observations are proposed.     

Kinetics and mechanism of the reduction of CrVI and CrV by D-lactobionic acid

The oxidation of D-lactobionic acid by Cr(VI) yields the 2-ketoaldobionic acid and Cr(3+) as final products when a 20-times or higher excess of the aldobionic acid over Cr(VI) is used. The redox reaction takes place through a complex multistep mechanism, which involves the formation of intermediate Cr(IV) and Cr(V) species. Cr(IV) reacts with lactobionic acid much faster than Cr(V) and Cr(VI) do, and cannot be directly detected. However, the formation of CrO(2)(2+), observed by the first time for an acid saccharide/Cr(VI) system, provides indirect evidence for the intermediacy of Cr(IV) in the reaction path. Cr(VI) and the intermediate Cr(V) react with lactobionic acid at comparable rates, being the complete rate laws for the Cr(VI) and Cr(V) consumption expressed by: -d[Cr(VI)]/dt=[k(I)+k(II)[H(+)]][lactobionicacid][Cr(VI)], where k(I)=(4.1+/-0.1) x 10(-3) M(-1) s(-1) and k(II)=(2.1+/-0.1) x 10(-2) M(-2) s(-1); and -d[Cr(V)]/dt=[k(III)[H(+)]+(k(IV)+k(V)[H(+)])[lactobionicacid]] [Cr(V)], where k(III)=(1.8+/-0.1) x 10(-3) M(-1) s(-1), k(IV)=(1.1+/-0.1) x 10(-2) M(-1) s(-1) and k(V)=(1.0+/-0.1) x 10(-2) M(-2) s(-1), at 33 degrees C. The Electron Paramagnetic Resonance (EPR) spectra show that five-co-ordinate oxo-Cr(V) bischelates are formed at pH 1-5 with the aldobionic acid bound to Cr(V) through the alpha-hydroxyacid group.     

Hydrophobic modulation of heme properties in heme protein maquettes

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.     

Probing site specificity of DNA binding metallointercalators by NMR spectroscopy and molecular modeling

The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.