- Email: [email protected]
Abstracts Poster Presentations (Z5)
Journal of Inorganic Biochemistry 86 (2001)
411
Heterologous expression of a ligninolytic versatile peroxidase from Pleurotus eryngii in Aspergillus species Francisco J. Ruiz-Duefias ~, Thelmo A. L6 Chau b, Marta Pfrez-Boada ~, Maria J. Martinez~, Angel T. Martinez a "Centro de Investigaciones Biol6gicas, Consejo Superior de Investigaciones Cientlficas, Veldzquez 144, 28006 Madrid, SPAIN (e-mail. [email protected]) '~'Department of Chemical Engineering, Institute of Technology, Av das Ciencias s/n, 15706 Santiago de Compostela, SPAIN A new versatile hemeperoxidase (VP), combining catalytic properties of LiP and MnP, has been described in fungi of genus Pleurotus. Gene vpl encoding a VP isoenzyme has been expressed in two different Aspergillus species under control of alcohol dehydrogenase (alcA) promoter. Firstly, expression in Emericella nidulans was performed and the effect of culture pH was studied. Increasing pH to 8.3 in no buffered cultures resulted in alkaline inactivation of recombinant VPL (VPL*). VPL reactivation by ~t .~.~,'1 adding CaC12 and dropping pH has been achieved in vitro, and expression optimization by C~I ii controlling pH in bioreactor. Peroxidase activity levels increased 5.3 times when compared ~~'~' ',~%1~;~, with those obtained without pH control, but they never surpassed 150 U/1. Then, genetically~ :~, modified Aspergillus niger Tgpdl (containing the codifying DNA sequence for the regulatory IE F::7 ~ :~-~:~s protein alcR) was chosen to improve the extracellular VPL* levels. Several recombinants ~ ~ ' ~_/
wereselectedandcheckedforperoxidaseactivityafterAnigertransformationwiththe expression vector pALMP2 ~. Cultures buffered with 100 mM sodium citrate at pH 5.5 yielded 740 U/l, almost the same activity levels (1000 U/l) obtained from P. e~ngii, OpUmization of' expression will be continued in bioreactor to improve VPL* production in A.
~...7~i r-~_~..._ ..... ~--,
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i. Ruiz-Duefias F. J., Martinez M.J. and Martinez A.T., A., Appl. Environ. Microbiol., 65, 4705-07 (1999)
C o m p l e x compounds of lanthanoides with macrocyclic ligands Adina Rus a, Ticuta Negreanu-Pirjol b, Leonid Carpus c, Elena Gorun d, Cornelia Guran a Department of Inorganic Chemistry, University Politehnica of Bucharest, Polizu 1,78126, Bucharest, Romania Faculty of Medicine and Pharmacy, OVIDIUS University, Ioan Voda 58, 8700, Constanta, Romania, e-mail. [email protected], ro • Public Health Institute, Constanta, Romania ,1 City Clinic Hospital, 8700, Constanta, Romania Proflavine is a synthetic dye that early in this century was found to have bacteriostatic and bactericidal properties when administered topically. The effect of proflavine, an antiseptic drug, on the spectroscopic and oxygen-binding properties of ferrous human adult haemoglobin (Hb) has been investigated I. In anticancer treatment is recently studied the effect of Ln ions 2. This paper presents the synthesis and characterisation of mono- and polynuclear complex compounds of Ce(III) with 3,6-diaminoacridine. Means of elemental chemical analysis, molar electric conductivity, electronic, AA and IR spectra established the formulae of the complexes. The antibacterial action of these complex compounds was confirmed by their action against Corynaebacterium diphteriae, Streptococcus haemolyticus group A., Streptococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa ser.VI, Pseudomonas aeruginosa ser.IX, Escherichia coli, Salmonella group B, Shigella flexneri, Yersinia Enterocolitica and Candida albicans. 1. 2.
Ascenzi P.,Colasanti M., Fasano M., Bertollini A., Biochem. Mol. Biol. Int., 47(6), 991-5, (1999) Kui Wang, Rongchang Li, Yi Cheng, Bing Zhu, Coord. Chem. Rev., 190-192, 297-308, (1999).
412
Journal of Inorganic Biochemistry 86 (2001)
Spectroscopic and photodynamic studies some of novel ytterbium-porphyrins Mari,/a Rusakova, Zinaida Zhilina, Sergey Vodzinskii, Tatyana Philippova 1.1. Mechnikov National University of Odessa, 2 Dvorianskaya str., 65026 Odessa, UKRAINE (e-mail: niv@ tm.odessa.ua) The synthesis of novel porphyrins and metalloporphyrins and related conjugated macrocycles continues to be a central part of intense investigations as to the potential use of these compounds as photosensitizers in bio-medical applications, such as viral inhibitor and photodynamic tumor therapy. The results of spectral-luminescent investigations for a number of solutions and solid-phase complexes of some ytterbium porphyrin complexes (free bases with aromatic substituents in meso-positions) are given in this paper. The synthesis of ligands (porphyrins) and complexes are considered in details.' Ligands and complexes have been characterized by elemental analysis, mass spectrometry, NMR-,IR-,UV-VIS absorption and luminescent spectroscopy. 4f-luminescence of ytterbium ions observed in the near-IR spectral region (emission maximum at 980 nm), where background of the bio-object radiation is practically absent. Thus, the best signal/noise rano can be obtained. A comparative study of the efficiency of luminescence excitation of Yb(III) ions and molecular portion of porphyrin complexes in liposomes and aqueous solutions (H20 and D20) has been made. The effects observed are associated with both possible monomerization of complexes in phosphatidylcholine liposomes and processes of radiationless energy. The lifetime of the metastable level of Yb(III) ions in porphyrin complexes is found to be dependent on their aggregate states and on the number of positions of nitrogen atoms in meso-substituents. The conditions under which the luminescence quantum yield is the largest have been defined. Also the photodynamic activity of these complexes was investigated by means of test-system, such as Saccharomyces cerevisial. The data obtained can help in selecting the most promising complexes for luminescent diagnostics of tumors in vitro and in vivo. 1. Philippova T., Galkin B., Golovenko N., Zhilina Z. and Vodzinskii S., J. Porphyrins Phtalocyanines, 4, 243-247 (2000) The Ministry of Education of Ukraine is acknowledged for financial support.
Study regarding the possible synthesis of bismuth complexes with clarithromycin Alice Rusu and Iulian Rusu Department of Inorganic Chemistry, Technical University of Iasi, Bd. D, Mangeron 71,6600, lasi, Romania,(e-maiI. [email protected]) It is well known that the bismuth compounds have been largely used for the treatment of different affections for more than two hundred years. On the other hand the clarithromycin is a last generation macrolide used with spectacular results for the treatment of gastric ulcer. This work is focused on the comparison between the therapeutic activity of clarithromycin and of a series of bismuth compounds on gastro-intestinal diseases. Furthermore, the complexation possibility of the macrolide with the Bi 3+ cation, in order to obtain a synergetic effect, is also taken into account. The analysis regards the synthesis in aqueous solution and in solid state by means of mechano-chemical activation. The solid state reactivity is to be analysed using the thermal analysis (TG, DTA, DSC), XRD and IR spectroscopy. The authors would like to acknowledge the generous aid granted by the Organizing Committee of ICBICIO in order to attend the conference.
Journal of Inorganic Biochemistry 86 (2001)
413
Heme-containing peptides as models for peroxidases Ekaterina S. Ryabovaa, Alexander Dikiyb, Stefano Ciurlib and Ebbe Nordlander a ~'Inorganic Chemistry l,Chemical Ceter Lund University, Sweden (e-mail: Ekaterina.Ryabova@inorg. lu.se) bDepartment of Agro-EnvironmentaI Science and Technology, University of Bologna, Italy. In order to create a heme environment which permits biomimicry of heme-peroxidases, a number of new hemepeptide complexes - hernin-2(18)-glycyl-L-histidine methyl ester (HGH), hemin-2(18)glycyl-glycyl-L-histidine methyl ester (HGGH) and hemin-2,18-bis(glycyl-glycyl-Lhistidine) dimethyl ester (H2GGH) - have been obtained by condensation of glycyl-L. ~7 ---q + histidine methyl ester and glycyl-glycyl-L-bistidine methyl ester on the propionic side ~c //~.,,~,.~ chains of heroin. Characterization by means of UV/vis- and ~H NMR-spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of fivex'~_ ." "-~ coordinate complexes in the case of HGH and HGGH with histidine as an axial ligand. In ~c --the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the : ~ ) ' ¢ / " . o~.H iron center appears to be formed; however, ~H NMR of H2GGH reveals the existence of N.~.~o an equilibrium between low-spin six-coordinate and high-spin five-coordinated species in solution. The catalytic activity of the heme-peptide complexes towards several organic substrates, such as p-cresol, L-tyrosine methyl ester and ABTS, has been investigated. It was found that not only the fivecoordinate HGH and HGGH, but also the six-coordinate H2GGH complex catalyze the oxidation by H202. The longer, and therefore less strained, peptide ann provides the HGGH complex with a slightly higher catalytic efficiency, compared to HGH, in peroxidative oxidation due to formation of more stable intermediate complexes. Formation of Compound I analogues was detected for all heme-peptide complexes: a shift of the Soret band and approx. 60% decrease of absorbance was observed.
Infrared spectroelectrochemistry of iron porphyrin nitrosyl complexes Michael D. Ryan, Hongqiang Zhang and Zhongcheng Wei Chemistry Department of Marquette University, Marquette University, PO Box 1881, Milwaukee, WI USA Iron porphyrin nitrosyls can be reduced in two one-eleclxon steps. The combination of electrochemistry and vibrational spectroscopy can provide information on the structure of the reduced complexes. The infrared spectroelectrochemistry of the iron porphyrin nitrosyl complexes were examined using both natural abundance nitrogen and lSN. The v(NO) band for the nitrosyl vibration 007 decreased from 1672 cm -t to 1437 cm ~ upon reduction. Nitrogen-15 isotopic shifts were consistent with this assignment. In the presence of a weak acid such as dichlorophenol (DCP), changes were observed in the reduction product, as shown in the figure to the right. Specifically, the 4307 absorbance for the reduced nitrosyl vibration decreased significantly in the presence of the weak acid, indicating further reduction of the complex. The ~0 structure of the product in the presence of weak acids was interpreted in 11900 1700 1500 1300 Wavenumber, crh light of the infrared spectroelectrochemical data, as well as the pulse polarographic data, which have been published earlier from this laboratory.
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414
Journal of Inorganic Biochemistry 86 (2001)
Peroxide activation by dinuclear and mononuclear iron model complexes: which intermediates are reactive? Elena V. Rybak-Akimova, Sergey V. Kryatov, Aida M. Herrera Department of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA 02155 USA (e-mail. [email protected]) Low temperature kinetic studies of formation and reactivity of peroxo- and high-valent oxo intermediates from synthetic models of non-heme iron oxidases and oxygenases are reported. A novel transient peroxo intermediate was found in the reaction of dinuclear Fe(III)-tris-picolylamine (TPA) complex with H202- i This intermediate reacts with a second molecule of H202, yielding the previously characterized '- diamond core +DTBP (le) +PhjP (2e) lZe(IIl)Fe(IV) species. Both dinuclear intermediates are kinetically competent in oxidation of substrate (triphenyl phosphine or 2,4-dik ~104 M'ls'i i tertbutylphenol, DTBP), as demonstrated by double-mixing stopped flow 0 0 0 /,,,\ ,,, experiments. The novel peroxo intermediate is far more reactive with the LF~"~o'"L-.zo~+.,o, D LFe Fe L k = 10]~I Is I \/ i I k,., = o.s s ~ I I substrates than the high valent diamond-core species (see Scheme). In O OH OH t OH~ OOH L= contrast, the mononuclear end-on peroxo intermediate (TPA)Fem(OOH) TPA does not oxidize DTBP and slowly reacts with triphenylphosphine. The l +DTBP (1 e) o r + Ph3P mechanisms of peroxo intermediate formation in several mononuclear k - 80 M'ts I k ~ 40 M'IS "l iron complexes with polyamine and aminopyridine ligands will also be discussed. 1. Kryatov S.V., Rybak-Akimova E . V . J . Chem. Soc. Dalton Trans., 3335-3336 (1999). 2. Que L., Jr. J. Chem. Soc. Dalton Trans., 3933-3940 (1997). This work was supported by Tufts University and the Research Corporation (RI 0223).
Ferrocenyl-nucleosides: synthesis, redox chemistry and incorporation into DNA Lyndsey Ryder, Andrew Pike, Benjamin Horrocks, Andrew Houlton, Department o f Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, NEI 7RU UNITED KINGDOM (e-mail: [email protected]) Attaching redox-active groups to sites in DNA is of considerable interest as these compounds may act as electrochemical probes for sensing applications, and can be used to address questions of DNA-mediated electron-transfer. Generally, such groups are appended as intact metal complexes after oligonucleotide synthesis to reactive terminal sites on the DNA strand. An alternative strategy, only recently considered, is to develop metallo-modified nucleosides which are compatible with solid phase oligonucleotide synthesis, i Towards this we have been investigating the application of ferrocenyl-nucleosides as electron-donor substituents in modified DNA and have prepared a series containing both conjugated and non-conjugated linkages. For thymidine, the C5-modified compound F c - ~ - d T , has been synthesised by Pd-catalysed cross-coupling. From (1) the related vinyl-, FcCH-CHdT (2), and ethyl-, FcCH2CH2dT (3), derivatives have been prepared by reduction. The synthesis, structural and electrochemical data of the compounds are presented along with the results of DNA incorporation. 1.
For example, Yu, et al., Ji Am. Chem. Soc. (2000) 122, 6767-6768. Hurley and Tor, J. Am. Chem. Soc., 1998, 120, 2194. Beilstein, Tierney, Grinstaff,. Commentslnorg. Chem. (2000), 22, 105.
The EPSRC is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
415
U n u s u a l stability of the heme-peroxidase from leaves of palm tree
Elaies guineensus I.Yu. Sakharov Department of Chemical Enzymology, Faculty of Chemistry, M. V.Lomonosov Moscow State University, Moscow, 119899, Russia (e-mail. [email protected]) Recently the novel peroxidase has been isolated to homogeneity using leaves of African oil palm tree Elaies guineensus as a source. Its molecular weight and isoelectric point values were 57.000 Da and 3.8, respectively. The palm anionic peroxidase (PAP) has a electronic UV-VIS spectrum characteristic for other plant peroxidases. PAP is stable in a broad range ofpH. The pH-stability was maximal at pH 7.0. Under neutral conditions PAP does not loss the activity at 80 °C for 1 h as a minimum. Moreover, PAP does not loss its activity in the presence of guanidinium hydrochloride up to concentration of 4 M at 25 °C. Under acidic and alkaline conditions the peroxidase showed less stability, but is stayed highly stable enzyme. So, PAP losses only not more than 20% of initial at pH 2.0 or 12.0 for 30 min, at 25 °C. According to the data about high stability of PAP under neutral pHs, the thermal inactivation of PAP has been studied at pH 2.2 and 10.5 at temperatures up to 84 °C. Under these conditions the addition of EDTA destabilizes PAP. However, DTT destabilizes PAP in significantly more extent than EDTA. Thus, disulfide bonds are likely to be crucial structural elements defined the unusual stability of PAP. PAP has also a high chemical stability. It was shown that the PAP is more resistant against organic solvents than horseradish peroxidase (HRP). Its activity in 40-50% of dioxane, ethanol and dimethylsulfoxide (common solvents in organic synthesis) was 3-10 fold than that of HRP. At the study of reaction of luminol oxidation by PAP the stable chemoluminescence signal has been registered for 1.5 h as a minimum. By the constant, HRP is chemically inactivated by the products of this reaction and therefore, in the case of HRP the signal value in the course of the reaction is changed constantly. The uniquely high stability detected permits us to hope about good biotechnological perspectives of PAP. This work was supported by the grants LST.CLG 975189 and 976273 of NATO Scientific Programme.
R a m a n spectroscopy approach to studies of enzyme conformation: activation of ~ - c h y m o t r y p s i n in organic media by crown ether I. K. Sakodinskayaa, a N. N. Brandtb,b A. Yu. Chikishev b Department of Chemistry, Moscow State University, Vorob'evy gory, Moscow. 119899 Russia; e-mail: [email protected] b internationai Laser Center and Physics Department, Moscow State University, Vorob'evy gory, Moscow, 119899 Russia Organic solvents are nowadays known to have a number of advantages for performing enzyme-catalysed- reactions, though the introduction of organic solvent results usually in a drop of enzymatic activity. Hence, an obvious problem is to increase the ability of enzymes to function in organic solvents with low water content. Crown ethers are known to activate serine proteases, tyrosinase and lipases in organic media: the catalytic activity of chymotrypsin in organic solvents is increased by several orders of magnitude in the presence of 18-crown-6. The possible mechanisms of catalytic activity amplification by crown ether in organic media are discussed and referred to the structural data. Functional activity of enzymes is known to be related to their conformational dynamics. Therefore, the knowledge of the conformational changes of chymotrypsin upon its interaction with different solvents and complex formation with crown ether is important for understanding of the mechanisms of the catalytic activity. In this work we used Raman spectroscopy for the study of the changes of chymotrypsin conformation caused by interactions with organic solvents and crown ether. The interaction of amino groups with crown ether was studied for the model compound tris(hydroxymethyl)aminomethane. We measured Raman spectra of chymotrypsin, tris, and the samples, pretreated with 18crown-6 at relative molar concentrations 1 : 10, 1:50, and 1 : 100 in powders, and suspended in acetonitrile and cyclohexane. The results are compared to the spectra of the enzyme in aqueous medium. The differences introduced by (i) organic solvent and by (ii) crown ether into the conformation of enzyme - a) secondary structure, b) tryptophane and c) tyrosine state and d) S-S bridges configuration are discussed and referred to the observed enzymatic activity. Complexation with the amino groups of the enzyme may be of crucial importance for the observed activation effects.
416
Journal of Inorganic Biochemistry 86 (2001)
Structure-activity relationship and action mechanism of insulinomimetic vanadylpieolinate complexes H. Sakurai a, K. K a w a b e a, H. Yasuia,y. K o j i m a b , y . Y o s h i k a w a b OKyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan (e-maiL [email protected]) bOsaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan The number of patients suffering from diabetes mellitus (DM) is increasing day by day. Type 1 DM can be controlled only by daily painful injections of insulin. Thus the development of compounds that cause insulin replacement or insulin action enhancing activity on oral administration is an urgent task in 21 century. Since our finding in 1995 that bis(picolinato)oxovanadinm(IV) complex (VO(pic)z) with a coordination mode of VO(N202) normalizes the high blood glucose levels of streptozotocin (STZ)-induced type 1 diabetic rats (STZ-rats) t), we have developed several types of vanadyl complexes by using VO(pic)2 as a leading complex. In designing the complexes, electron donating or electron withdrawing substituents were introduced to the picolinate ligand. In vitro insulinomimetic activity of the prepared complexes was evaluated by inhibition of free fatty acids release from isolated rat adipocytes stimulated with epinephrine, in terms of IC50 value, which expresses 50% inhibitory concentration of each complex. Among them, introductions of methyl group at 3 or 6 position and iodine at 5 position of the picolinate ligand were found to give higher insulinomimetic activities than the leading complex. These three complexes exhibited good antidiabetic blood glucose normalizing effects on STZ-rats when they were given daily by ip injection or oral administration. To understand the action mechanism of such vanadyl complexes, we examined the action site of vanadyl ion and complexes in isolated rat adipocytes by using several types of inhibitors, and found that both vanadyl ion and complexes act on multiple sites including glucose transporter in the insulin-induced signal transduction pathways. Reference: 1) Sakurai, H., Fujii, K., Watanabe, H., Tamura, H., Biochem. Biophys. Res. Commun., 214, 1095-1101 (1995)
Structural and functional mechanisms ofDesulfovibrio vulgaris cytochrome c3 revealed by site specific mutagenesis Carlos A. Salgueiro a'b, Patr/cia N. da Costa a, David L. T u m e r a'c, A n a C. Messias a, Walter M.A.M. van D o n g e n d, M . L i g i a M. Saraiva", and Ant6nio V. X a v i e r a
~ITQB.-UNL, Apt. 127, 2780-156 Oeiras, Portugal (e-mail. [email protected] bDepartamento Quimica, FCT-UNL, Quinta da Torre, 2825-114 Caparica, Portugal CDepartment of Chemistry, University of Southampton, Southampton S 0 1 7 1B J, UK d Department of Biochemistry, Wageningen Agricultural University, Wageningen, The Netherlands The structural basis for the redox properties of the tetrahaem Desulfovibrio vulgaris cytochrome c3, has been investigated by site specific mutagenesis. Thermodynamic and structural characterisation of mutated cytochromes revealed the functional role of Lys45 and Thr24 in the wild type protein. Mutation of Lys45, located in the vicinity of the propionates of haem I, by the uncharged residues Thr and Gin, allowed to conclude that Lys45 controls the pKa of such propionate groups and confirms the involvement of this residue in the redox-Bohr effect~. More recently, the replacement of Thr24, located in the vicinity ofhaem III, by Val showed how crucial Thr24 is for the stabilisation of the oxidised form of the protein. This single mutation increases the microscopic redox potential ofhaem III by 106 mV. This mutation also destabilizes the concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild type protein. Therefore, Thr24 is responsible for the balance of the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer2. 1. Saraiva, L.M., Salgueiro, C.A., da Costa, P.N., Messias, A.C., LeGall, J., van Dongen, W.M.A.M. and Xavier, A.V., Biochemistry 37, 12160-12165 (1998). 2. Salgueiro, C.A., da Costa, P.N., Turner, D.L. Messias, A.C,, van Dongen, W.M.A.M, Saraiva, L.M. and Xavier, A.V., Biochemistry (2001) in press.
Journal of lnorganic Biochemistry 86 (2001)
417
Photochemical consequences of sunscreen physical and chemicalfilters of UVA/UVB sunlight radiation on DNA plasmids, human cells and yeast cells Angela Salinaro a, John Knowland b, Nick Serpone a " Department of Chemistry and Biochemistry, Concordia University, Montreal (Quebec), Canada H3G 1M8.( e-mail: [email protected]) b Department of Biochemistry, Oxford University, South Parks Road, Oxford OXI 3QU, UK Sunscreen lotions are used to prevent the immediate effects on skin exposed to harmful UVA (320-400 nm) and UVB (290-320 nm) sunlight, and to prevent skin cancers (carcinomas and melanoma). The incidence of skin cancers continues to rise worldwide at an alarming rate despite massive education and availability of suncreams. UVB sunlight causes sunburns (erythema) and damage to skin. Topical sunscreens should provide a protective layer of exogeneous UV chromophores on the skin to block UV radiation before it penetrates the epidermis and affect DNA, so that photochemistry and subsequent photobiology are reduced. Sunscreens that penetrate the epidermis and the cells, if irradiated, yield photoadducts and/or generate reactive oxyradicals that cause DNA lesions. The effects are felt several years later as premature aged skin and as skin melanoma. UVA sunlight also contributes to actinic premature skin aging and skin cancer. Sunscreens that block UVB radiation only may enhance skin damage. Manufacturers use TiO2 and ZnO as effective broadband physicalfilters. However, the titanium dioxide currently used in micronized form in most sunblock lotions absorbs appreciable UV radiation and produces QOH radicals that cause considerable damage to DNA. Sunlight illuminated TiO2 catalyzes DNA damage in vitro, in human cells, and in vivo in yeast cells. (ZnO behaves stmilarly). The photoactive nature of these metal oxides requires changes to the particle surface to suppress formation of oxyradicals and thereby suppress DNA damage as evidenced by nicking assays, comet assays, and survival of yeast cells. Several titania specimens were passivated; they display substantial reduction of photoactivity such that they significantly reduce damage to DNA, and by extrapolation to human skin. Most important, the broadband UVA/UVB protection by titania specimens is improved (or unaltered) by surface passivation. We also show that popular chemicalfilters are not photochemically stable; their UV protective features are questionable. Our work is supported by NSERC Canada to NS.
Mechanistic look at the orthoplatination of aryl oximes by dichlorobissulfoxide platinum(II) complexes Pavel V. Samuleev, Alexander D. Ryabov, Vladirnir A. Polyakov, Grigory M. Kazankov Department of Chemistry, Lomonosov's Moscow State University, Vorob'evi Gori, MGU, 119899, Moscow, Russia (e-mail. [email protected]) The platinum(II) sulfoxide metalacycles has recently attracted considerable attention. They have been shown to be promising for creation of centers with the planar chirality, for the modeling of biologically relevant systems such as DMSO reductase, and for the efficient catalysis of hydrolysis of pesticides of the parathion family. Broad potential applications of these compounds preserve substantial interest in further exploring the synthetic capacity of the Pt H sulfoxide complexes in preparation of platinacycles via direct C-H bond cleavage. Here we report the structural and mechanistic aspects of orthoplatination of acetophenone oxime by the platinum(II) sulfoxide + [NCI~L2} ----1~ + HC1 + L complexes [PtClzL2 ] (2, L - SOMe2 (a), rac-SOMePh (b), RSOMe(C6H4Me-4) (c)) to afford the corresponding platinacycles N.~O H 2 L--P~ I N~'OH cis(C,S)-[PtH(C6H4-Z-CCH3=NOH)CI(L)] (3). l Ol It was shown that the orthoplatination is a multistep process. At the first step the formation of unreactive chlorobis(N-oxime)(SSOMez)platinum(II) cations accounts for the rate retardation by excess of acetophenone oxime and suggests the importance of pseudo-coordinatively unsaturated species for the C-H bond activation by Ptjl. Second step is independent of the oxime concentration indicating that it is intramolecular in nature. The latter observation suggests that it could involve the C-H bond cleavage to form the platinacycle. The INTAS is acknowledged for fmancial support. (grant No 97-0166)
418
Journal of Inorganic Biochemistry 86 (2001)
EPR evidence for an S=7/2 intermediate state from the Mn4 cluster of photosystem II Yiannis Sanakis a' b Nikos Ioannidis ~, George Sioros ~, Vasili Petrouleas a " Inst. of Mat, Sci. NCSR "Demokritos", 15310 Ag. Paraskevi, Attiki, Greece h Department of Biological Applications and Technologies, University ofloannina,loannina, Greece (e-mail: [email protected]) Water oxidation in Photosystem II (PSII) takes place at a mixed valence cluster comprising 4 Mn atoms. EPR spectroscopy has been used extensively to the study of the complex. A recent advance is = 4.2K /,~ the detection of bimodal EPR signals from the integer-spin $3 state.l' 2 These signals change drastically upon NIR light excitation at l.He temperatures, z Notable is the formation of an EPR signal comprising a broad (AHpp-22.0 mT) gaussian derivative with zero crossing point at g-4.80. Interestingly enough a similar signal is obtained following prolonged dark incubation (days) of the $3 state at 77K, 3 (figure). We describe this signal within the context of the usual spin-Hamiltonian H = D[&2-S(S+I)/3]+E(S~:-Sy2)+goflBS. 800 1200 1600 2000 A detailed analysis indicates that the signal arises from the [+3/2> doublet of an S=7/2 B (a) state. Depending on the conditions of formation, the zero field splitting parameters vary. When the signal is formed after prolonged incubation at 77K the ZFS parameters are D1=0.55 cm 1 and E/D-0.11. The I.R induced signal is characterized by IDI-2.0 cm -~ and E/D-0.10, and this probably reflects the influence of the nearby tyr Z. Unresolved hyperfme interactions with the 55Mn nuclei account for the line broadening. We attribute the S=7/2 signal to a modified (proton-deficient) $2 state of the Mn4 cluster of PSII. This is the first detailed characterization of a high spin state with S>5/2 from the mixed valence Mn4 cluster of PSI[. 1. Matsukawa,T., Mino, H., Yoneda, D. and Kawamori, A., Biochemistry, 38, 4072-4077 (1999) 2. Ioannidis, N. and Petrouleas, V. Biochemistry, 39, 5246-5254 (2000) 3. Nugent, J.H.A., Turconi, S. and Evans, M.C.W., Biochemistry, 36, 7086-7096 (1997)
~
Carbon monoxide binding by coprinus cinereus peroxidase and selected mutants Elisa Santoni a, Alessandro Feis a, Karen G. Welinder b, and Giulietta Smulevich a Dipartimento di Chimica, Universita' di Firenze, Via G. Capponi 9, 1-50121 Firenze, Italy (e-mail: elisa@chim, unifi, it) ~' lnstitut for Bioteknoigi, Aalborg Universitet, Sohngaardsholmsvej 49, DK-9000 Aalborg, Dennlark. The CO complexes of Coprinus cinereus peroxidase (CIP) and of selected mutants have been investigated using IR and resonance Raman spectroscopies to obtain information on the heme cavity architecture. The study of CO complexes of the Arg51 mutants (Arg51Leu, Arg51Gln, Arg51Asn, and Arg5 I Lys) demonstrates that while Arg51 does not bind CO, at variance with cytochrome c (CCP) ~and horseradish (HRPC) 2 peroxidases, it plays an important role in enabling CO to assume a position which facilitates H-bonding to the distal His. Moreover, the substitution of Arg with Leu weakens the proximal Fe-His bond, demonstrating the strong link between the proximal and the distal heme cavities. The CO complexes of the Phe54 mutants (Phe54Trp and Phe54Val) show that also this residue, like Arg5 I, does not bind CO, but facilitates H-bonding to distal His. In the case of the proximal cavity, the results confirm that substitution of Asp245 with Asn weakens the proximal Fe-His bond as a consequence of the weakening of the Asp245-His proximal H-bonding. These results point to a larger distal heme cavity for CIP as compared to CCP and HRPC. 1. 2.
Smulevich G., Mauro M.J., Fishel L.A., English A.M., Kraut J. and Spiro T.G., Biochemistry 27, 5486-5492 (1988) Feis A., Rodriguez-Lopez J.N., Thorneley R.N.F. and Smulevich G., Biochemistry 39, 13575-13581 (1998)
The EU (contract BIO4-97-2031) and the Italian Ministero delrUniversita' e Ricerca Scientifica e Tecnologica (MURST) are acknowledged for financial support.
Journal o f lnorganic Biochemistry 86 (2001)
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A new hexadentate 3-hydroxy-4-pyridinonate ligand as a chelator for iron gallium and aluminium. In vitro and in vivo studies M. A m d l i a S a n t o s a, R a q u e l G r a z i n a a, L u r d e s G a n o u, G u i l h e r m i n a C a n t i n o c P o r t u g a l ; C e n t r o de Q u i m i c a E s t r u t u r a l , C o m p l e x o 1, Instituto S u p e r i o r T~cnico, 1 0 4 9 - 0 0 1 Lisboa, c I n s t i t u t o T e c n o l 6 g i c o e N u c l e a r , E s t r a d a N a c i o n a l N ° 10, 2 6 8 6 - 9 5 3 S a c a v e m , P o r t u g a l , h [ n s t i t u t o de M e d i c i n a N u c l e a r , F a c u l d a d e de M e d i c i n a de Lisboa, R. E g a s Monis, Lisboa, Portugal The development of new chelators for trivalent ions, such as iron, gallium and aluminium has been the object of current interest due to their potential clinical use, either in radiodiagnosis (67'68Ga) or in chelating therapy of A1 or Fe overload patients. LAs part of an ongoing project we have developed a new tris(hydroxypyridinonate) ligand and we report herein the results of some in vitro and in vivo studies. The set of the model-studies includes the results of evaluation of its binding affinity towards those metal ions, in aqueous medium, and of partition coefficients (octanol/water) at physiological pH for the ligand and the corresponding complexes, Very stable and water-soluble complexes are formed with a trend on its binding efficiency according to the order Ga>Fe>AI. Biodistribution of 67Ga-citrate was studied in mice with and without simultaneous injection of the chelator, as previouly reported by us for a set ofbidentate chelators. Data from both studies was compared and led us to conclude that this new hexadentate ligand interferes in the normal biological profile of 67Ga-citrate enhancing the excretion of 67Ga, although considerable differences were detected at the level of organ uptakes. l. R. A Yokel, A. M. Fredenburg, P. W. Durbin, J, Xu, M. K. Rayens, K. N. Raymond, J. Pharm.Sci. 89 (2000) 545-555. The authors thank to the Portuguese Funda~do para a Ci6ncia e Tecnologia (FCT) (project POCTI/35368/QUI/2000).
N-benzyl- iminodiacetohydroxamate and carboxymethylhydroxamate ligands. Synthesis and complexation studies with Cu(II), Ni(II), Zn(II) M. Amdlia Santos, Sergio Marques, Marco Gil, Silvia Chaves Centro de Quimica Estrutural, Instituto Superior T~cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Hydroxamm acids and amino-hydroxamic acids are known to play an important role in living systems. Recently, they have been extensively studied in the field of the zinc and nickel metalloenzyme inhibition. ~ This set ofhydroxamic acids have an imino-diacetate backbone skeleton with one or two hydroxamate chelating units. On the other hand, one N-benzyl group is inserted to simulate the lipophilic subset on the interaction with enzyme proteins. We report herein the synthetic procedures to obtain these ligands and their complexation behaviours towards the transition metal ions (Cu 2+, Ni 2+ and Zn 2+) in aqueous solution, which were studied by pH-metric and UV-Vis spectrophotometric techniques. Their preparation was based on the derivatization of the corresponding iminodiacetic acid by standard methods. The equilibrium studies indicated that both the ligands form quite stable water-soluble complexes and they mostly behave like a-aminohydroxamic acids. The binding modes showed dependence on the pit, namely changing from {N,N}amine-hydroxamate to {O,O} hydroxamate co-ordination with increasing pH. The eventual existence of polynuclear species with mixed coordination is also admitted. The results are discussed in comparison with similar analogues having larger spacers between the chelating moities, previously reported by us. 2 1. M.H. Parker, E. A. Lunney, D. F. Ortwine, A. G. Pavlovsky, C. Humblet and C. G. Brouillette, Biochemistry, 1999, 38, 13592-13601. 2. M.A. Santos, R. Grazina, M. Pinto and E. Farkas, Inorg, Chim. Acta, in publication. The authors thank to the COST D21 program and to the Portuguese Foundation for Science and Technology.
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Journal of Inorganic Biochemistry 86 (2001)
Chaperone-assisted heterologous expression and purification of an active nitrile hydratase (Nil) from comamonas testosteroni Marie-Agnbs Sail a, Julie M. Stevens a, M a y a Belghazi b, M a r y s e Jaouen a, Jean-Marie Sctunitter b, Didier B o n n e t a, Daniel M a n s u y a, Isabelle Artaud a aLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR8601, UniversitO Paris V, 45 rue des Saints-POres, 75006Paris, FRANCE ([email protected]). b bTstitut EuropOen de Chimie et de Biologie, Eeole Polytechnique, Universit~s de Bordeaux 1 et II, B.P. 108, Talence, FRANCE
Nitrile hydratases (Nhases) are industrially significant enzymes used in the large-scale synthesis of acrylamide. These enzymes contain either non-heme iron or non-corrinoid cobalt ions in their active sites. Previous reports have shown that the active site peptides of Nhases are post-translationally modified by cysteine oxidation(1), and that these modifications are essential for catalysis. Attempts to express recombinant iron Nhases have been unsuccessfull unless specific "activators " are coexpressed (2). We report a heterologous system for the expression of functionnal recombinant ironcontaining Nhase in E coli. The system involves the co-expression of the two Nhase subunits with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be similar to the original Comamonas enzyme in terms of its catalytic and spectroscopic properties. Mass spectrometric evidence of the oxidation of the active site cysteines will be presented for the full-length recombinant and the native Nil Nhase. 1. Nagashima S., Nakasako M., Tsujimura M., Odaka K., Yohda M., Kamiya N. and Endo I., Nature Struct. Biol., 5, 347-351 (1998) 2. Nojiri M., Yohda M., Odaka M., Matsuhita Y., Tsujimura M., Yoshida T., Dohmae N., Takio K., Endo I., J. Biochem., 125,696-704 (1999) This work was funded by the European Commission TMR-NOHEMIP research network ERBFMRXCT98-0174.
Photo-induced ligand migration and small moleculeactivation using biologically important metal centers D o n a l d V. Scaltrito, H. Christopher Fry, David W. T h o m p s o n , H o n g C h a n g - L i a n g , Kenneth D. Karlin, and Gerald J. M e y e r Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA (email: [email protected]) Synthetic model systems of cytochrome c oxidase, CcO, have been prepared and photo-induced reactions with small molecules investigated. It has been shown using time-resolved absorption spectroscopy that carbon monoxide, CO, can photo-dissociate from the individual heine Fe(II) and Cu(I) centers in fluid media. To our knowledge, the observation of CO photo-dissociation in Cu(I) compounds is unprecedented. Quantum yields for CO loss and the kinetics for CO rebinding have been quantified and the results indicate that small molecule activation can be photo-initiated in Cu(I)carbonyl compounds.~ This strategy has been extended to investigate CO migration from the heine Fe(II) center to the Cu(I) center in CcO model systems. Photo-induced ligand migration has proven to be a powerful tool for probing how Fe(II) and Cu(I) metal centers interact with biologically relevant small molecules. 1. Scaltrito, D.V., Fry, H.C., Showalter, B.M., Thompson, D.W., Liang, H.C., Zhang, C,X., Kim, E., Toscano, J.P., Karlin, K.D., and Meyer, G.J., submitted for publication. The Petroleum Research Foundation and the National Institutes of Health are gratefully acknowledged for financial support of this work.
411
Heterologous expression of a ligninolytic versatile peroxidase from Pleurotus eryngii in Aspergillus species Francisco J. Ruiz-Duefias ~, Thelmo A. L6 Chau b, Marta Pfrez-Boada ~, Maria J. Martinez~, Angel T. Martinez a "Centro de Investigaciones Biol6gicas, Consejo Superior de Investigaciones Cientlficas, Veldzquez 144, 28006 Madrid, SPAIN (e-mail. [email protected]) '~'Department of Chemical Engineering, Institute of Technology, Av das Ciencias s/n, 15706 Santiago de Compostela, SPAIN A new versatile hemeperoxidase (VP), combining catalytic properties of LiP and MnP, has been described in fungi of genus Pleurotus. Gene vpl encoding a VP isoenzyme has been expressed in two different Aspergillus species under control of alcohol dehydrogenase (alcA) promoter. Firstly, expression in Emericella nidulans was performed and the effect of culture pH was studied. Increasing pH to 8.3 in no buffered cultures resulted in alkaline inactivation of recombinant VPL (VPL*). VPL reactivation by ~t .~.~,'1 adding CaC12 and dropping pH has been achieved in vitro, and expression optimization by C~I ii controlling pH in bioreactor. Peroxidase activity levels increased 5.3 times when compared ~~'~' ',~%1~;~, with those obtained without pH control, but they never surpassed 150 U/1. Then, genetically~ :~, modified Aspergillus niger Tgpdl (containing the codifying DNA sequence for the regulatory IE F::7 ~ :~-~:~s protein alcR) was chosen to improve the extracellular VPL* levels. Several recombinants ~ ~ ' ~_/
wereselectedandcheckedforperoxidaseactivityafterAnigertransformationwiththe expression vector pALMP2 ~. Cultures buffered with 100 mM sodium citrate at pH 5.5 yielded 740 U/l, almost the same activity levels (1000 U/l) obtained from P. e~ngii, OpUmization of' expression will be continued in bioreactor to improve VPL* production in A.
~...7~i r-~_~..._ ..... ~--,
tiger
i. Ruiz-Duefias F. J., Martinez M.J. and Martinez A.T., A., Appl. Environ. Microbiol., 65, 4705-07 (1999)
C o m p l e x compounds of lanthanoides with macrocyclic ligands Adina Rus a, Ticuta Negreanu-Pirjol b, Leonid Carpus c, Elena Gorun d, Cornelia Guran a Department of Inorganic Chemistry, University Politehnica of Bucharest, Polizu 1,78126, Bucharest, Romania Faculty of Medicine and Pharmacy, OVIDIUS University, Ioan Voda 58, 8700, Constanta, Romania, e-mail. [email protected], ro • Public Health Institute, Constanta, Romania ,1 City Clinic Hospital, 8700, Constanta, Romania Proflavine is a synthetic dye that early in this century was found to have bacteriostatic and bactericidal properties when administered topically. The effect of proflavine, an antiseptic drug, on the spectroscopic and oxygen-binding properties of ferrous human adult haemoglobin (Hb) has been investigated I. In anticancer treatment is recently studied the effect of Ln ions 2. This paper presents the synthesis and characterisation of mono- and polynuclear complex compounds of Ce(III) with 3,6-diaminoacridine. Means of elemental chemical analysis, molar electric conductivity, electronic, AA and IR spectra established the formulae of the complexes. The antibacterial action of these complex compounds was confirmed by their action against Corynaebacterium diphteriae, Streptococcus haemolyticus group A., Streptococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa ser.VI, Pseudomonas aeruginosa ser.IX, Escherichia coli, Salmonella group B, Shigella flexneri, Yersinia Enterocolitica and Candida albicans. 1. 2.
Ascenzi P.,Colasanti M., Fasano M., Bertollini A., Biochem. Mol. Biol. Int., 47(6), 991-5, (1999) Kui Wang, Rongchang Li, Yi Cheng, Bing Zhu, Coord. Chem. Rev., 190-192, 297-308, (1999).
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Journal of Inorganic Biochemistry 86 (2001)
Spectroscopic and photodynamic studies some of novel ytterbium-porphyrins Mari,/a Rusakova, Zinaida Zhilina, Sergey Vodzinskii, Tatyana Philippova 1.1. Mechnikov National University of Odessa, 2 Dvorianskaya str., 65026 Odessa, UKRAINE (e-mail: niv@ tm.odessa.ua) The synthesis of novel porphyrins and metalloporphyrins and related conjugated macrocycles continues to be a central part of intense investigations as to the potential use of these compounds as photosensitizers in bio-medical applications, such as viral inhibitor and photodynamic tumor therapy. The results of spectral-luminescent investigations for a number of solutions and solid-phase complexes of some ytterbium porphyrin complexes (free bases with aromatic substituents in meso-positions) are given in this paper. The synthesis of ligands (porphyrins) and complexes are considered in details.' Ligands and complexes have been characterized by elemental analysis, mass spectrometry, NMR-,IR-,UV-VIS absorption and luminescent spectroscopy. 4f-luminescence of ytterbium ions observed in the near-IR spectral region (emission maximum at 980 nm), where background of the bio-object radiation is practically absent. Thus, the best signal/noise rano can be obtained. A comparative study of the efficiency of luminescence excitation of Yb(III) ions and molecular portion of porphyrin complexes in liposomes and aqueous solutions (H20 and D20) has been made. The effects observed are associated with both possible monomerization of complexes in phosphatidylcholine liposomes and processes of radiationless energy. The lifetime of the metastable level of Yb(III) ions in porphyrin complexes is found to be dependent on their aggregate states and on the number of positions of nitrogen atoms in meso-substituents. The conditions under which the luminescence quantum yield is the largest have been defined. Also the photodynamic activity of these complexes was investigated by means of test-system, such as Saccharomyces cerevisial. The data obtained can help in selecting the most promising complexes for luminescent diagnostics of tumors in vitro and in vivo. 1. Philippova T., Galkin B., Golovenko N., Zhilina Z. and Vodzinskii S., J. Porphyrins Phtalocyanines, 4, 243-247 (2000) The Ministry of Education of Ukraine is acknowledged for financial support.
Study regarding the possible synthesis of bismuth complexes with clarithromycin Alice Rusu and Iulian Rusu Department of Inorganic Chemistry, Technical University of Iasi, Bd. D, Mangeron 71,6600, lasi, Romania,(e-maiI. [email protected]) It is well known that the bismuth compounds have been largely used for the treatment of different affections for more than two hundred years. On the other hand the clarithromycin is a last generation macrolide used with spectacular results for the treatment of gastric ulcer. This work is focused on the comparison between the therapeutic activity of clarithromycin and of a series of bismuth compounds on gastro-intestinal diseases. Furthermore, the complexation possibility of the macrolide with the Bi 3+ cation, in order to obtain a synergetic effect, is also taken into account. The analysis regards the synthesis in aqueous solution and in solid state by means of mechano-chemical activation. The solid state reactivity is to be analysed using the thermal analysis (TG, DTA, DSC), XRD and IR spectroscopy. The authors would like to acknowledge the generous aid granted by the Organizing Committee of ICBICIO in order to attend the conference.
Journal of Inorganic Biochemistry 86 (2001)
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Heme-containing peptides as models for peroxidases Ekaterina S. Ryabovaa, Alexander Dikiyb, Stefano Ciurlib and Ebbe Nordlander a ~'Inorganic Chemistry l,Chemical Ceter Lund University, Sweden (e-mail: Ekaterina.Ryabova@inorg. lu.se) bDepartment of Agro-EnvironmentaI Science and Technology, University of Bologna, Italy. In order to create a heme environment which permits biomimicry of heme-peroxidases, a number of new hemepeptide complexes - hernin-2(18)-glycyl-L-histidine methyl ester (HGH), hemin-2(18)glycyl-glycyl-L-histidine methyl ester (HGGH) and hemin-2,18-bis(glycyl-glycyl-Lhistidine) dimethyl ester (H2GGH) - have been obtained by condensation of glycyl-L. ~7 ---q + histidine methyl ester and glycyl-glycyl-L-bistidine methyl ester on the propionic side ~c //~.,,~,.~ chains of heroin. Characterization by means of UV/vis- and ~H NMR-spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of fivex'~_ ." "-~ coordinate complexes in the case of HGH and HGGH with histidine as an axial ligand. In ~c --the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the : ~ ) ' ¢ / " . o~.H iron center appears to be formed; however, ~H NMR of H2GGH reveals the existence of N.~.~o an equilibrium between low-spin six-coordinate and high-spin five-coordinated species in solution. The catalytic activity of the heme-peptide complexes towards several organic substrates, such as p-cresol, L-tyrosine methyl ester and ABTS, has been investigated. It was found that not only the fivecoordinate HGH and HGGH, but also the six-coordinate H2GGH complex catalyze the oxidation by H202. The longer, and therefore less strained, peptide ann provides the HGGH complex with a slightly higher catalytic efficiency, compared to HGH, in peroxidative oxidation due to formation of more stable intermediate complexes. Formation of Compound I analogues was detected for all heme-peptide complexes: a shift of the Soret band and approx. 60% decrease of absorbance was observed.
Infrared spectroelectrochemistry of iron porphyrin nitrosyl complexes Michael D. Ryan, Hongqiang Zhang and Zhongcheng Wei Chemistry Department of Marquette University, Marquette University, PO Box 1881, Milwaukee, WI USA Iron porphyrin nitrosyls can be reduced in two one-eleclxon steps. The combination of electrochemistry and vibrational spectroscopy can provide information on the structure of the reduced complexes. The infrared spectroelectrochemistry of the iron porphyrin nitrosyl complexes were examined using both natural abundance nitrogen and lSN. The v(NO) band for the nitrosyl vibration 007 decreased from 1672 cm -t to 1437 cm ~ upon reduction. Nitrogen-15 isotopic shifts were consistent with this assignment. In the presence of a weak acid such as dichlorophenol (DCP), changes were observed in the reduction product, as shown in the figure to the right. Specifically, the 4307 absorbance for the reduced nitrosyl vibration decreased significantly in the presence of the weak acid, indicating further reduction of the complex. The ~0 structure of the product in the presence of weak acids was interpreted in 11900 1700 1500 1300 Wavenumber, crh light of the infrared spectroelectrochemical data, as well as the pulse polarographic data, which have been published earlier from this laboratory.
°-@
414
Journal of Inorganic Biochemistry 86 (2001)
Peroxide activation by dinuclear and mononuclear iron model complexes: which intermediates are reactive? Elena V. Rybak-Akimova, Sergey V. Kryatov, Aida M. Herrera Department of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA 02155 USA (e-mail. [email protected]) Low temperature kinetic studies of formation and reactivity of peroxo- and high-valent oxo intermediates from synthetic models of non-heme iron oxidases and oxygenases are reported. A novel transient peroxo intermediate was found in the reaction of dinuclear Fe(III)-tris-picolylamine (TPA) complex with H202- i This intermediate reacts with a second molecule of H202, yielding the previously characterized '- diamond core +DTBP (le) +PhjP (2e) lZe(IIl)Fe(IV) species. Both dinuclear intermediates are kinetically competent in oxidation of substrate (triphenyl phosphine or 2,4-dik ~104 M'ls'i i tertbutylphenol, DTBP), as demonstrated by double-mixing stopped flow 0 0 0 /,,,\ ,,, experiments. The novel peroxo intermediate is far more reactive with the LF~"~o'"L-.zo~+.,o, D LFe Fe L k = 10]~I Is I \/ i I k,., = o.s s ~ I I substrates than the high valent diamond-core species (see Scheme). In O OH OH t OH~ OOH L= contrast, the mononuclear end-on peroxo intermediate (TPA)Fem(OOH) TPA does not oxidize DTBP and slowly reacts with triphenylphosphine. The l +DTBP (1 e) o r + Ph3P mechanisms of peroxo intermediate formation in several mononuclear k - 80 M'ts I k ~ 40 M'IS "l iron complexes with polyamine and aminopyridine ligands will also be discussed. 1. Kryatov S.V., Rybak-Akimova E . V . J . Chem. Soc. Dalton Trans., 3335-3336 (1999). 2. Que L., Jr. J. Chem. Soc. Dalton Trans., 3933-3940 (1997). This work was supported by Tufts University and the Research Corporation (RI 0223).
Ferrocenyl-nucleosides: synthesis, redox chemistry and incorporation into DNA Lyndsey Ryder, Andrew Pike, Benjamin Horrocks, Andrew Houlton, Department o f Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, NEI 7RU UNITED KINGDOM (e-mail: [email protected]) Attaching redox-active groups to sites in DNA is of considerable interest as these compounds may act as electrochemical probes for sensing applications, and can be used to address questions of DNA-mediated electron-transfer. Generally, such groups are appended as intact metal complexes after oligonucleotide synthesis to reactive terminal sites on the DNA strand. An alternative strategy, only recently considered, is to develop metallo-modified nucleosides which are compatible with solid phase oligonucleotide synthesis, i Towards this we have been investigating the application of ferrocenyl-nucleosides as electron-donor substituents in modified DNA and have prepared a series containing both conjugated and non-conjugated linkages. For thymidine, the C5-modified compound F c - ~ - d T , has been synthesised by Pd-catalysed cross-coupling. From (1) the related vinyl-, FcCH-CHdT (2), and ethyl-, FcCH2CH2dT (3), derivatives have been prepared by reduction. The synthesis, structural and electrochemical data of the compounds are presented along with the results of DNA incorporation. 1.
For example, Yu, et al., Ji Am. Chem. Soc. (2000) 122, 6767-6768. Hurley and Tor, J. Am. Chem. Soc., 1998, 120, 2194. Beilstein, Tierney, Grinstaff,. Commentslnorg. Chem. (2000), 22, 105.
The EPSRC is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
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U n u s u a l stability of the heme-peroxidase from leaves of palm tree
Elaies guineensus I.Yu. Sakharov Department of Chemical Enzymology, Faculty of Chemistry, M. V.Lomonosov Moscow State University, Moscow, 119899, Russia (e-mail. [email protected]) Recently the novel peroxidase has been isolated to homogeneity using leaves of African oil palm tree Elaies guineensus as a source. Its molecular weight and isoelectric point values were 57.000 Da and 3.8, respectively. The palm anionic peroxidase (PAP) has a electronic UV-VIS spectrum characteristic for other plant peroxidases. PAP is stable in a broad range ofpH. The pH-stability was maximal at pH 7.0. Under neutral conditions PAP does not loss the activity at 80 °C for 1 h as a minimum. Moreover, PAP does not loss its activity in the presence of guanidinium hydrochloride up to concentration of 4 M at 25 °C. Under acidic and alkaline conditions the peroxidase showed less stability, but is stayed highly stable enzyme. So, PAP losses only not more than 20% of initial at pH 2.0 or 12.0 for 30 min, at 25 °C. According to the data about high stability of PAP under neutral pHs, the thermal inactivation of PAP has been studied at pH 2.2 and 10.5 at temperatures up to 84 °C. Under these conditions the addition of EDTA destabilizes PAP. However, DTT destabilizes PAP in significantly more extent than EDTA. Thus, disulfide bonds are likely to be crucial structural elements defined the unusual stability of PAP. PAP has also a high chemical stability. It was shown that the PAP is more resistant against organic solvents than horseradish peroxidase (HRP). Its activity in 40-50% of dioxane, ethanol and dimethylsulfoxide (common solvents in organic synthesis) was 3-10 fold than that of HRP. At the study of reaction of luminol oxidation by PAP the stable chemoluminescence signal has been registered for 1.5 h as a minimum. By the constant, HRP is chemically inactivated by the products of this reaction and therefore, in the case of HRP the signal value in the course of the reaction is changed constantly. The uniquely high stability detected permits us to hope about good biotechnological perspectives of PAP. This work was supported by the grants LST.CLG 975189 and 976273 of NATO Scientific Programme.
R a m a n spectroscopy approach to studies of enzyme conformation: activation of ~ - c h y m o t r y p s i n in organic media by crown ether I. K. Sakodinskayaa, a N. N. Brandtb,b A. Yu. Chikishev b Department of Chemistry, Moscow State University, Vorob'evy gory, Moscow. 119899 Russia; e-mail: [email protected] b internationai Laser Center and Physics Department, Moscow State University, Vorob'evy gory, Moscow, 119899 Russia Organic solvents are nowadays known to have a number of advantages for performing enzyme-catalysed- reactions, though the introduction of organic solvent results usually in a drop of enzymatic activity. Hence, an obvious problem is to increase the ability of enzymes to function in organic solvents with low water content. Crown ethers are known to activate serine proteases, tyrosinase and lipases in organic media: the catalytic activity of chymotrypsin in organic solvents is increased by several orders of magnitude in the presence of 18-crown-6. The possible mechanisms of catalytic activity amplification by crown ether in organic media are discussed and referred to the structural data. Functional activity of enzymes is known to be related to their conformational dynamics. Therefore, the knowledge of the conformational changes of chymotrypsin upon its interaction with different solvents and complex formation with crown ether is important for understanding of the mechanisms of the catalytic activity. In this work we used Raman spectroscopy for the study of the changes of chymotrypsin conformation caused by interactions with organic solvents and crown ether. The interaction of amino groups with crown ether was studied for the model compound tris(hydroxymethyl)aminomethane. We measured Raman spectra of chymotrypsin, tris, and the samples, pretreated with 18crown-6 at relative molar concentrations 1 : 10, 1:50, and 1 : 100 in powders, and suspended in acetonitrile and cyclohexane. The results are compared to the spectra of the enzyme in aqueous medium. The differences introduced by (i) organic solvent and by (ii) crown ether into the conformation of enzyme - a) secondary structure, b) tryptophane and c) tyrosine state and d) S-S bridges configuration are discussed and referred to the observed enzymatic activity. Complexation with the amino groups of the enzyme may be of crucial importance for the observed activation effects.
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Structure-activity relationship and action mechanism of insulinomimetic vanadylpieolinate complexes H. Sakurai a, K. K a w a b e a, H. Yasuia,y. K o j i m a b , y . Y o s h i k a w a b OKyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan (e-maiL [email protected]) bOsaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan The number of patients suffering from diabetes mellitus (DM) is increasing day by day. Type 1 DM can be controlled only by daily painful injections of insulin. Thus the development of compounds that cause insulin replacement or insulin action enhancing activity on oral administration is an urgent task in 21 century. Since our finding in 1995 that bis(picolinato)oxovanadinm(IV) complex (VO(pic)z) with a coordination mode of VO(N202) normalizes the high blood glucose levels of streptozotocin (STZ)-induced type 1 diabetic rats (STZ-rats) t), we have developed several types of vanadyl complexes by using VO(pic)2 as a leading complex. In designing the complexes, electron donating or electron withdrawing substituents were introduced to the picolinate ligand. In vitro insulinomimetic activity of the prepared complexes was evaluated by inhibition of free fatty acids release from isolated rat adipocytes stimulated with epinephrine, in terms of IC50 value, which expresses 50% inhibitory concentration of each complex. Among them, introductions of methyl group at 3 or 6 position and iodine at 5 position of the picolinate ligand were found to give higher insulinomimetic activities than the leading complex. These three complexes exhibited good antidiabetic blood glucose normalizing effects on STZ-rats when they were given daily by ip injection or oral administration. To understand the action mechanism of such vanadyl complexes, we examined the action site of vanadyl ion and complexes in isolated rat adipocytes by using several types of inhibitors, and found that both vanadyl ion and complexes act on multiple sites including glucose transporter in the insulin-induced signal transduction pathways. Reference: 1) Sakurai, H., Fujii, K., Watanabe, H., Tamura, H., Biochem. Biophys. Res. Commun., 214, 1095-1101 (1995)
Structural and functional mechanisms ofDesulfovibrio vulgaris cytochrome c3 revealed by site specific mutagenesis Carlos A. Salgueiro a'b, Patr/cia N. da Costa a, David L. T u m e r a'c, A n a C. Messias a, Walter M.A.M. van D o n g e n d, M . L i g i a M. Saraiva", and Ant6nio V. X a v i e r a
~ITQB.-UNL, Apt. 127, 2780-156 Oeiras, Portugal (e-mail. [email protected] bDepartamento Quimica, FCT-UNL, Quinta da Torre, 2825-114 Caparica, Portugal CDepartment of Chemistry, University of Southampton, Southampton S 0 1 7 1B J, UK d Department of Biochemistry, Wageningen Agricultural University, Wageningen, The Netherlands The structural basis for the redox properties of the tetrahaem Desulfovibrio vulgaris cytochrome c3, has been investigated by site specific mutagenesis. Thermodynamic and structural characterisation of mutated cytochromes revealed the functional role of Lys45 and Thr24 in the wild type protein. Mutation of Lys45, located in the vicinity of the propionates of haem I, by the uncharged residues Thr and Gin, allowed to conclude that Lys45 controls the pKa of such propionate groups and confirms the involvement of this residue in the redox-Bohr effect~. More recently, the replacement of Thr24, located in the vicinity ofhaem III, by Val showed how crucial Thr24 is for the stabilisation of the oxidised form of the protein. This single mutation increases the microscopic redox potential ofhaem III by 106 mV. This mutation also destabilizes the concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild type protein. Therefore, Thr24 is responsible for the balance of the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer2. 1. Saraiva, L.M., Salgueiro, C.A., da Costa, P.N., Messias, A.C., LeGall, J., van Dongen, W.M.A.M. and Xavier, A.V., Biochemistry 37, 12160-12165 (1998). 2. Salgueiro, C.A., da Costa, P.N., Turner, D.L. Messias, A.C,, van Dongen, W.M.A.M, Saraiva, L.M. and Xavier, A.V., Biochemistry (2001) in press.
Journal of lnorganic Biochemistry 86 (2001)
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Photochemical consequences of sunscreen physical and chemicalfilters of UVA/UVB sunlight radiation on DNA plasmids, human cells and yeast cells Angela Salinaro a, John Knowland b, Nick Serpone a " Department of Chemistry and Biochemistry, Concordia University, Montreal (Quebec), Canada H3G 1M8.( e-mail: [email protected]) b Department of Biochemistry, Oxford University, South Parks Road, Oxford OXI 3QU, UK Sunscreen lotions are used to prevent the immediate effects on skin exposed to harmful UVA (320-400 nm) and UVB (290-320 nm) sunlight, and to prevent skin cancers (carcinomas and melanoma). The incidence of skin cancers continues to rise worldwide at an alarming rate despite massive education and availability of suncreams. UVB sunlight causes sunburns (erythema) and damage to skin. Topical sunscreens should provide a protective layer of exogeneous UV chromophores on the skin to block UV radiation before it penetrates the epidermis and affect DNA, so that photochemistry and subsequent photobiology are reduced. Sunscreens that penetrate the epidermis and the cells, if irradiated, yield photoadducts and/or generate reactive oxyradicals that cause DNA lesions. The effects are felt several years later as premature aged skin and as skin melanoma. UVA sunlight also contributes to actinic premature skin aging and skin cancer. Sunscreens that block UVB radiation only may enhance skin damage. Manufacturers use TiO2 and ZnO as effective broadband physicalfilters. However, the titanium dioxide currently used in micronized form in most sunblock lotions absorbs appreciable UV radiation and produces QOH radicals that cause considerable damage to DNA. Sunlight illuminated TiO2 catalyzes DNA damage in vitro, in human cells, and in vivo in yeast cells. (ZnO behaves stmilarly). The photoactive nature of these metal oxides requires changes to the particle surface to suppress formation of oxyradicals and thereby suppress DNA damage as evidenced by nicking assays, comet assays, and survival of yeast cells. Several titania specimens were passivated; they display substantial reduction of photoactivity such that they significantly reduce damage to DNA, and by extrapolation to human skin. Most important, the broadband UVA/UVB protection by titania specimens is improved (or unaltered) by surface passivation. We also show that popular chemicalfilters are not photochemically stable; their UV protective features are questionable. Our work is supported by NSERC Canada to NS.
Mechanistic look at the orthoplatination of aryl oximes by dichlorobissulfoxide platinum(II) complexes Pavel V. Samuleev, Alexander D. Ryabov, Vladirnir A. Polyakov, Grigory M. Kazankov Department of Chemistry, Lomonosov's Moscow State University, Vorob'evi Gori, MGU, 119899, Moscow, Russia (e-mail. [email protected]) The platinum(II) sulfoxide metalacycles has recently attracted considerable attention. They have been shown to be promising for creation of centers with the planar chirality, for the modeling of biologically relevant systems such as DMSO reductase, and for the efficient catalysis of hydrolysis of pesticides of the parathion family. Broad potential applications of these compounds preserve substantial interest in further exploring the synthetic capacity of the Pt H sulfoxide complexes in preparation of platinacycles via direct C-H bond cleavage. Here we report the structural and mechanistic aspects of orthoplatination of acetophenone oxime by the platinum(II) sulfoxide + [NCI~L2} ----1~ + HC1 + L complexes [PtClzL2 ] (2, L - SOMe2 (a), rac-SOMePh (b), RSOMe(C6H4Me-4) (c)) to afford the corresponding platinacycles N.~O H 2 L--P~ I N~'OH cis(C,S)-[PtH(C6H4-Z-CCH3=NOH)CI(L)] (3). l Ol It was shown that the orthoplatination is a multistep process. At the first step the formation of unreactive chlorobis(N-oxime)(SSOMez)platinum(II) cations accounts for the rate retardation by excess of acetophenone oxime and suggests the importance of pseudo-coordinatively unsaturated species for the C-H bond activation by Ptjl. Second step is independent of the oxime concentration indicating that it is intramolecular in nature. The latter observation suggests that it could involve the C-H bond cleavage to form the platinacycle. The INTAS is acknowledged for fmancial support. (grant No 97-0166)
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Journal of Inorganic Biochemistry 86 (2001)
EPR evidence for an S=7/2 intermediate state from the Mn4 cluster of photosystem II Yiannis Sanakis a' b Nikos Ioannidis ~, George Sioros ~, Vasili Petrouleas a " Inst. of Mat, Sci. NCSR "Demokritos", 15310 Ag. Paraskevi, Attiki, Greece h Department of Biological Applications and Technologies, University ofloannina,loannina, Greece (e-mail: [email protected]) Water oxidation in Photosystem II (PSII) takes place at a mixed valence cluster comprising 4 Mn atoms. EPR spectroscopy has been used extensively to the study of the complex. A recent advance is = 4.2K /,~ the detection of bimodal EPR signals from the integer-spin $3 state.l' 2 These signals change drastically upon NIR light excitation at l.He temperatures, z Notable is the formation of an EPR signal comprising a broad (AHpp-22.0 mT) gaussian derivative with zero crossing point at g-4.80. Interestingly enough a similar signal is obtained following prolonged dark incubation (days) of the $3 state at 77K, 3 (figure). We describe this signal within the context of the usual spin-Hamiltonian H = D[&2-S(S+I)/3]+E(S~:-Sy2)+goflBS. 800 1200 1600 2000 A detailed analysis indicates that the signal arises from the [+3/2> doublet of an S=7/2 B (a) state. Depending on the conditions of formation, the zero field splitting parameters vary. When the signal is formed after prolonged incubation at 77K the ZFS parameters are D1=0.55 cm 1 and E/D-0.11. The I.R induced signal is characterized by IDI-2.0 cm -~ and E/D-0.10, and this probably reflects the influence of the nearby tyr Z. Unresolved hyperfme interactions with the 55Mn nuclei account for the line broadening. We attribute the S=7/2 signal to a modified (proton-deficient) $2 state of the Mn4 cluster of PSII. This is the first detailed characterization of a high spin state with S>5/2 from the mixed valence Mn4 cluster of PSI[. 1. Matsukawa,T., Mino, H., Yoneda, D. and Kawamori, A., Biochemistry, 38, 4072-4077 (1999) 2. Ioannidis, N. and Petrouleas, V. Biochemistry, 39, 5246-5254 (2000) 3. Nugent, J.H.A., Turconi, S. and Evans, M.C.W., Biochemistry, 36, 7086-7096 (1997)
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Carbon monoxide binding by coprinus cinereus peroxidase and selected mutants Elisa Santoni a, Alessandro Feis a, Karen G. Welinder b, and Giulietta Smulevich a Dipartimento di Chimica, Universita' di Firenze, Via G. Capponi 9, 1-50121 Firenze, Italy (e-mail: elisa@chim, unifi, it) ~' lnstitut for Bioteknoigi, Aalborg Universitet, Sohngaardsholmsvej 49, DK-9000 Aalborg, Dennlark. The CO complexes of Coprinus cinereus peroxidase (CIP) and of selected mutants have been investigated using IR and resonance Raman spectroscopies to obtain information on the heme cavity architecture. The study of CO complexes of the Arg51 mutants (Arg51Leu, Arg51Gln, Arg51Asn, and Arg5 I Lys) demonstrates that while Arg51 does not bind CO, at variance with cytochrome c (CCP) ~and horseradish (HRPC) 2 peroxidases, it plays an important role in enabling CO to assume a position which facilitates H-bonding to the distal His. Moreover, the substitution of Arg with Leu weakens the proximal Fe-His bond, demonstrating the strong link between the proximal and the distal heme cavities. The CO complexes of the Phe54 mutants (Phe54Trp and Phe54Val) show that also this residue, like Arg5 I, does not bind CO, but facilitates H-bonding to distal His. In the case of the proximal cavity, the results confirm that substitution of Asp245 with Asn weakens the proximal Fe-His bond as a consequence of the weakening of the Asp245-His proximal H-bonding. These results point to a larger distal heme cavity for CIP as compared to CCP and HRPC. 1. 2.
Smulevich G., Mauro M.J., Fishel L.A., English A.M., Kraut J. and Spiro T.G., Biochemistry 27, 5486-5492 (1988) Feis A., Rodriguez-Lopez J.N., Thorneley R.N.F. and Smulevich G., Biochemistry 39, 13575-13581 (1998)
The EU (contract BIO4-97-2031) and the Italian Ministero delrUniversita' e Ricerca Scientifica e Tecnologica (MURST) are acknowledged for financial support.
Journal o f lnorganic Biochemistry 86 (2001)
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A new hexadentate 3-hydroxy-4-pyridinonate ligand as a chelator for iron gallium and aluminium. In vitro and in vivo studies M. A m d l i a S a n t o s a, R a q u e l G r a z i n a a, L u r d e s G a n o u, G u i l h e r m i n a C a n t i n o c P o r t u g a l ; C e n t r o de Q u i m i c a E s t r u t u r a l , C o m p l e x o 1, Instituto S u p e r i o r T~cnico, 1 0 4 9 - 0 0 1 Lisboa, c I n s t i t u t o T e c n o l 6 g i c o e N u c l e a r , E s t r a d a N a c i o n a l N ° 10, 2 6 8 6 - 9 5 3 S a c a v e m , P o r t u g a l , h [ n s t i t u t o de M e d i c i n a N u c l e a r , F a c u l d a d e de M e d i c i n a de Lisboa, R. E g a s Monis, Lisboa, Portugal The development of new chelators for trivalent ions, such as iron, gallium and aluminium has been the object of current interest due to their potential clinical use, either in radiodiagnosis (67'68Ga) or in chelating therapy of A1 or Fe overload patients. LAs part of an ongoing project we have developed a new tris(hydroxypyridinonate) ligand and we report herein the results of some in vitro and in vivo studies. The set of the model-studies includes the results of evaluation of its binding affinity towards those metal ions, in aqueous medium, and of partition coefficients (octanol/water) at physiological pH for the ligand and the corresponding complexes, Very stable and water-soluble complexes are formed with a trend on its binding efficiency according to the order Ga>Fe>AI. Biodistribution of 67Ga-citrate was studied in mice with and without simultaneous injection of the chelator, as previouly reported by us for a set ofbidentate chelators. Data from both studies was compared and led us to conclude that this new hexadentate ligand interferes in the normal biological profile of 67Ga-citrate enhancing the excretion of 67Ga, although considerable differences were detected at the level of organ uptakes. l. R. A Yokel, A. M. Fredenburg, P. W. Durbin, J, Xu, M. K. Rayens, K. N. Raymond, J. Pharm.Sci. 89 (2000) 545-555. The authors thank to the Portuguese Funda~do para a Ci6ncia e Tecnologia (FCT) (project POCTI/35368/QUI/2000).
N-benzyl- iminodiacetohydroxamate and carboxymethylhydroxamate ligands. Synthesis and complexation studies with Cu(II), Ni(II), Zn(II) M. Amdlia Santos, Sergio Marques, Marco Gil, Silvia Chaves Centro de Quimica Estrutural, Instituto Superior T~cnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Hydroxamm acids and amino-hydroxamic acids are known to play an important role in living systems. Recently, they have been extensively studied in the field of the zinc and nickel metalloenzyme inhibition. ~ This set ofhydroxamic acids have an imino-diacetate backbone skeleton with one or two hydroxamate chelating units. On the other hand, one N-benzyl group is inserted to simulate the lipophilic subset on the interaction with enzyme proteins. We report herein the synthetic procedures to obtain these ligands and their complexation behaviours towards the transition metal ions (Cu 2+, Ni 2+ and Zn 2+) in aqueous solution, which were studied by pH-metric and UV-Vis spectrophotometric techniques. Their preparation was based on the derivatization of the corresponding iminodiacetic acid by standard methods. The equilibrium studies indicated that both the ligands form quite stable water-soluble complexes and they mostly behave like a-aminohydroxamic acids. The binding modes showed dependence on the pit, namely changing from {N,N}amine-hydroxamate to {O,O} hydroxamate co-ordination with increasing pH. The eventual existence of polynuclear species with mixed coordination is also admitted. The results are discussed in comparison with similar analogues having larger spacers between the chelating moities, previously reported by us. 2 1. M.H. Parker, E. A. Lunney, D. F. Ortwine, A. G. Pavlovsky, C. Humblet and C. G. Brouillette, Biochemistry, 1999, 38, 13592-13601. 2. M.A. Santos, R. Grazina, M. Pinto and E. Farkas, Inorg, Chim. Acta, in publication. The authors thank to the COST D21 program and to the Portuguese Foundation for Science and Technology.
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Journal of Inorganic Biochemistry 86 (2001)
Chaperone-assisted heterologous expression and purification of an active nitrile hydratase (Nil) from comamonas testosteroni Marie-Agnbs Sail a, Julie M. Stevens a, M a y a Belghazi b, M a r y s e Jaouen a, Jean-Marie Sctunitter b, Didier B o n n e t a, Daniel M a n s u y a, Isabelle Artaud a aLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR8601, UniversitO Paris V, 45 rue des Saints-POres, 75006Paris, FRANCE ([email protected]). b bTstitut EuropOen de Chimie et de Biologie, Eeole Polytechnique, Universit~s de Bordeaux 1 et II, B.P. 108, Talence, FRANCE
Nitrile hydratases (Nhases) are industrially significant enzymes used in the large-scale synthesis of acrylamide. These enzymes contain either non-heme iron or non-corrinoid cobalt ions in their active sites. Previous reports have shown that the active site peptides of Nhases are post-translationally modified by cysteine oxidation(1), and that these modifications are essential for catalysis. Attempts to express recombinant iron Nhases have been unsuccessfull unless specific "activators " are coexpressed (2). We report a heterologous system for the expression of functionnal recombinant ironcontaining Nhase in E coli. The system involves the co-expression of the two Nhase subunits with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be similar to the original Comamonas enzyme in terms of its catalytic and spectroscopic properties. Mass spectrometric evidence of the oxidation of the active site cysteines will be presented for the full-length recombinant and the native Nil Nhase. 1. Nagashima S., Nakasako M., Tsujimura M., Odaka K., Yohda M., Kamiya N. and Endo I., Nature Struct. Biol., 5, 347-351 (1998) 2. Nojiri M., Yohda M., Odaka M., Matsuhita Y., Tsujimura M., Yoshida T., Dohmae N., Takio K., Endo I., J. Biochem., 125,696-704 (1999) This work was funded by the European Commission TMR-NOHEMIP research network ERBFMRXCT98-0174.
Photo-induced ligand migration and small moleculeactivation using biologically important metal centers D o n a l d V. Scaltrito, H. Christopher Fry, David W. T h o m p s o n , H o n g C h a n g - L i a n g , Kenneth D. Karlin, and Gerald J. M e y e r Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA (email: [email protected]) Synthetic model systems of cytochrome c oxidase, CcO, have been prepared and photo-induced reactions with small molecules investigated. It has been shown using time-resolved absorption spectroscopy that carbon monoxide, CO, can photo-dissociate from the individual heine Fe(II) and Cu(I) centers in fluid media. To our knowledge, the observation of CO photo-dissociation in Cu(I) compounds is unprecedented. Quantum yields for CO loss and the kinetics for CO rebinding have been quantified and the results indicate that small molecule activation can be photo-initiated in Cu(I)carbonyl compounds.~ This strategy has been extended to investigate CO migration from the heine Fe(II) center to the Cu(I) center in CcO model systems. Photo-induced ligand migration has proven to be a powerful tool for probing how Fe(II) and Cu(I) metal centers interact with biologically relevant small molecules. 1. Scaltrito, D.V., Fry, H.C., Showalter, B.M., Thompson, D.W., Liang, H.C., Zhang, C,X., Kim, E., Toscano, J.P., Karlin, K.D., and Meyer, G.J., submitted for publication. The Petroleum Research Foundation and the National Institutes of Health are gratefully acknowledged for financial support of this work.