abstracts poster presentations (s)

Journal of Inorganic Biochemistry 86 (2001) 301

Radicals and solvated electrons generated by ionizing radiatio in the system: water-methanol-sulphonated metal phtalocyanines

M. Krowicka a, E. Kaleciflska a, J. K a l e c i f i s k i b

a F a c u l t y of Chemistry, University o f Wroclaw, 14 F. Joliot-Curie St,, 50-383 Wroclaw, Poland; b W. Trzebiatowski Institute o f Low Temperature and Structure Research, Polish Academy of Sciences, POBox 1410, 50-950 Wroclaw, Poland (e-mail. gosiak@wchuwr, chem. uni. wroc.pO

Metal phthalocyanines are known as the good chemicals applied in photodynamic therapy and possibly as the good radiosensitizers. Hence the knowledge of their behaviour under ionizing irradiation should be interesting.

In the present work the rate constants of reaction of metal tetrasulfophthalocyanines (Mtsp n-, M = n , 3+ ~ 2+ -~, 2+ ,.-, 2+ "2+ . . . . tvn , wo , ,vm , wu and N1 ) with e solv, OH and CH2OH radmals have been determmed. The rate contants of electron scavenging by Mtsp n- was measured by pulse radiolysis metod. The rate constants of OH scavenging by Mtsp n- have been measured in water solutions by competition method with pNDA (p-nitrosodimethylaniline) in gamma radiolysis experiments. In methanol-water solutions the rate constants of C H 2 O H radicals scavenging b y Mtsp n- are significantly lower than that of OH scavenging. High values of electron scavenging as well as OH radicals scavenging by Mtsp n- independently on the metals imply the essential role of nitrogen ring of phthalocyanine in these processes.

We acknowledge receipt of financial support from the KBN, Grant No 3T09A 168 18

Bis(aminomethyl)phosphinic acid - synthesis, acido-basic and complexing properties

Voitich Kubieek, Petr Hermann, Ivan Luke,, Jan Kotek, Pavel Vojtigek, lvana Cisa~ov~t

Department of Inorganic Chemistry, Charles University, Hlavova 8, 120 O0 Prague, Czech Republic, (e-mail: [email protected])

Bis(aminomethyl)phosphinic acid was synthetised by reaction of bis(trimethylsilyl)hypophosphite with N- brommethylphtalimide in high yield. The phtalimide protecting groups were removed by acid hydrolysis. The ligand was crystallized as a monohydrochloride and its structure was determined by X-ray diffraction.

Protonization constants of the ligand and stability constants of its complexes with Co >, Cu >, Ni 2+ and Zn 2+ ions were determined using potenciometric titrations. Values of stability 0~ ..¢ constants are higher than these for other aminomethylphosphinic acids ~. In case of systems .~, "

• 2 + with Cu 2÷ and Na , they are comparable with values found for aminocarboxylic and 0, aminophosphonic acids 2.

Complexes with Co 2+, Co 3., Cu 2+, Ni2+and Zn 2+ were prepared in the solid state. The Cu 2+ complex was microcrystallic, structure of the other complexes was determined by X- ray diffraction. All central ions show octahedral coordination sphere, but the ligand is coordinated in different ways.

1. Rohovec J., Lukeg I., Vojtigek P., Cisa~ov~ I and Hermann P., J. Chem. Soc.; Dalton Trans., 2685 (1996) 2. Martell A. E. and Smith R. M .... Critical Stability Constants ", Vols.l-6, Plenum Press, New York (1974-1989)

The Grant Agency of the Czech Republic (GACR) is acknowledged for a financial support.

302 Journal of Inorganic Biochemistry 86 (2001)

Key residues in amine oxidases and lysyl oxidation

Jason A. K u c h a r a and D a v i d M. D o o l e y a

a Department of Chemistry, Montana State University, 108 Gaines Hall, 59717, Bozeman MT, USA (e-mail.- uchddjk@gemini, oscs. montana, edu)

Two key residues in or near the active site of PPLO (copper containing amine oxidase from Pichia pastoris) have been mutated and characterized. The first was Tyr384 7 Phe. This tyrosine is absolutely conserved among amine oxidases and is hydrogen bonded to TPQ in the known X-ray structures. This mutant had a reduced specific activity and altered substrate specificity. The second mutant designed was Thr474 L_' Leu. This threonme is part of the active site consensus sequence (T-X-X-N-Y-D/E). TPQ formation and activity was greatly reduced m this mutant. The activity of various native amine oxidases versus peptidyl lysine residues was also investigated.

Acknowledgment: Eric Shepard, Greg Juda, and Bradley Elmore (Department of Chemistry, Montana State University, 108 Games Hall, 59717, Bozeman MT, USA).

Oxygenolysis of alkali metal catecholates in aprotic solvents

/ i . Kupf in a and G. Spe i e r b.

Department of Organic Chemistry, University of VeszprOm, 8201 Veszpr~m, HUNGARY (e-mail: [email protected])

b Research Group for Petrochemistry, Hungarian Academy of Sciences, 8201 Veszpr~m, H UNGA R Y

Many of the enzymes have a hydrophobic environment at their active center and therefore it seems reasonable to study model reactions in aprotic solvents. According to that we studied the oxygenation of alkali metal salts of 3,5-di- tert-butylcatechol, as functional model systems of 1,2-catecholdioxygenase.

The potassium and sodium salts of the catechols react with dioxygen under anaerob conditions in ether, THF and DMF in a fast reaction with the stoichiometry 1:1 leading first to the semiquinones and superoxide ion. The semiquinone reacts both with 02 and 02" in consecutive reactions resulting the alkali metal muconates as the ring cleaved products. In the first fast reaction step, one electron is transferred from catecholate to O2 giving semiquinones (EPR) and O2" These react than in slow, second order (rate-determining) step to the endproducts. The reaction was followed by spectrophotometry and gasvolumetry, the rate constants were determined according to the rate law -d[DTBSQM]/dt = k[DTBSQM][ 02"]. The reactions exhibit chemiluminescence indicating the transient presence of 1,2-dioxetane intermediates.

The Hungarian Research Found (OTKA T-030400) is acknowledged for financial support.package.


Ruthenium(II) complexed to poly-N-vinylimidazole as a mediator of electrical communicat ion between electrodes and active sites of

glucose oxidase or glucose dehydrogenase

V.S. K u r o v a , a A . D . R y a b o v , a W. S c h u h m a n n . b

M.V. Lomonosov Moscow State University, Department o f Chemistry, 119899, Moscow, Russia

Ruhr-Universitiit Bochum, Lehrstuhl fiir Analytishe Chemie, D-44780, Bochum, Germany

Ruthenium redox polymer based on poly-N-vinylimidazole (Ru-PVI) of the 4:1 polymer to metal ratio was synthesised and investigated as an electron mediator for oxidoreductases. The high redox potential of the polymer (570 mV vs Ag/AgCI at pH 7.0) does not allow to use it in glucose biosensors, but it can be a good model for studies of the electron transfer between the active site of glucose oxidase (GO) modified with cis-[Ru(bpy)2C12] and electrode. Native and modified enzymes were incorporated with Ru-PVI in a redox film cross-linked by epoxy cement on the surface of carbon electrode. The presence of Ru complexes in GO influence strongly the Michaelis constant KME and the effective electrochemical rate constant for the enzyme electrode kME, but not on the rate constant for transport of the substrate through the film (ks). The coordination of GO and the Ru complex leads to an increase both in the glucose response of the electrodes and the limiting catalytic current densityj,,~. Thus, the kc~t for the modified GO is two tunes higher compared to native enzyme when the GO contents is in range 22.6-36.8 wt %. At 22.6 and 36.8 wt % the jm~ is identical for modified and unmodified GO. These facts suggest a formation a sort of assembly between ruthenium centres of modified GO and Ru-PVI, which facilitates the electrocatalytic oxidation of glucose at the Ru-PVI enzyme electrodes.

We acknowledge support from INTAS (Project 99-166), Russian Foundation for Basic Research (99-03-34328a) and fellowship from the Ruhr Universit/it Bochum.

Structure of the 2-hydrazinopyridine adduct II of e. coli amine oxidase

C. Kurtis a, M. Mure b, D. Brown c, M. Rogers c, P. Knowles a, D. Dooley c, M. Parsons a, M. M c P h e r s o n a

" Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, Leeds, United Kingdom. ([email protected]. uk)

"Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

~Department o f Chemistry, and Biochemistry, Montana State University, Bozeman 59717, USA.

The amine oxidase from Escherichia coli (ECAO) is a dimeric, 160kDa copper-containing monoamine oxidase (EC 1.4.3.6). The mature form of the enzyme contains a modified tyrosine cofactor, trihydroxyphenylalanine quinone (TPQ) ~. The preferred substrate of this enzyme is phenylethylamine; at pH 7, Rear/Kin ~100 MI.s -1, however, the enzyme will also oxidase benzylamine; at pH 7 k~,t/Km -1 M t .s 1. The benzylamine analogue 2-Hydrazinopyridine (2-HP) is a suicide inhibitor of ECAO that forms a spectroscopically distinct TPQ adduct (kmax ~420nm) 2. Over time this adduci converts to a second, species (kmax -530nm). Formation of the second adduct is relatively slow in wild type ECAO, the rate of conversion being increased by raising the temperature and/or pH of the reaction. We have generated a variant form of ECAO, in which a conserved active site residue, Tyr369, has been changed to a phenylalanine (Y369F). Tyr369 is not critical for catalytic function; at pH7 k~at/Km -2.1 M'I.s 1. In Y369F, the rate of formation of adduct II is significantly increased; at pH 8.5 the majority of the adducted TPQ is adduct II form. The 2.3A resolution crystal structure of the adducted Y369F protein has been determined in Leeds. The structural, resonance Raman and model compound studies suggest that the TPQ and pyridine rings of adduct I are conjugated and that the observed red shift in the spectra of adduct II is attributable to the formation of a complex with the active site cupric ion. The rate of formation of adduct II in Y369F is explained by a diminution in the structurally-stabilising active site hydrogen bonding network in which the OH group of Tyr369 appears to play a key role.

1. Janes SM, Mu D, Wemmer D, Smith AJ, Kaur S, Maltby D, Burlingame AL, Klinman JP., Science, 248, 981- 987, 1990

2. Wilmot CM, Murray JM, Alton G, Parsons MR, Convery MA, Blakeley V, Comer AS, Palcic MM, Knowles PF, McPherson MJ, Phillips SE., Biochemistry, 36(7), 1608-1620, 1997


Separating the roles of homo- and heterosubunit interactions in the Rieske dioxygenases. Effects in anthranilate dioxygenase

Donald .M. Kurtz, Jr. a , Zanna M. Beharry a, D. Matthew Eby b, Eric D. Coulter a, Ellen L. Neidle b, Robert S. Phillips a

Department of Chemistry, Center for Metalloenzyme Studies and bDepartment of Microbiology University of Georgia, Athens, Georgia 30602, U.S.A. (email. [email protected])

The Rieske dioxygenase, anthranilate (2-aminobenzoate) 1,2-dioxygenase (AntDO) catalyzes insertion of 02 into the aromatic ring of anthranilate at a mononuclear non-heme iron site leading to catechol.~ Herein, we report mutation of aspartate 218, the residue proposed to bridge the Rieske and mononuclear sites in adjacent ~x subunits of the c~3133 hexameric AntDO to alanine (D218AAntDO) and characterization of the mutated enzyme. In addition, we have separately isolated and characterized the c~3 trimer of AntDO in an effort to understand the roles of the 13 subunit in the structure and function of AntDO. D218AantDO shows no oxygenase activity towards anthranilate nor any uncoupled oxidase activity, whereas AntA shows anthranilate-dependent, uncoupled oxidase activity. The bridging aspartate, D218, thus, appears to be absolutely required for electron flow from Rieske~mononuclear iron~dioxygen, whereas the [3 subunit appears to be required for coupling this electron flow to substrate oxygenation.

1. Eby, D.M., Beharry, Z.M., Coulter, E.D., Kurtz, D.M., Jr., and Neidle, E.L., J. Bacteriol., 183, 109-118 (2001)

This work was supported by National Institutes of Health grant GM 59818.

Palladacycles as catalysts of hydrolysis of S-containing amino acid derivatives

S.A. Kurzeev, G.M. Kazankov, A.D. Ryabov. Departament of Chemistry, M.V. Lomonosov Moscow State University, 119899, Moscow, Russia, (e-mail." sa [email protected])

Ortho-palladated primary and tertiary benzylamines catalyze the hydrolysis of sulfur containing amino acid esters. Kinetic studies suggest the intramolecular mechanism of the hydrolysis via the 6- or 7-membered transition states which involve the by Pd-S bond formation. Significant increase in the catalytic efficacy was observed (1) for primary benzylamines compared to the tertiary ones, (2) in the presence of substitutes at the c~ position of benzylamine, (3) when palladacycles contained Br, I-, O A c as acido ligands instead of CI-, (4) on decreasing the bulkiness of N-protecting group, and (5) on decreasing the bulkiness of the R radical of the amino acid RCH(NHz)COOH. It was concluded that the spatial orientation and hydrophobic interactions account for the level of the observed catalytic activity.


Solution N M R determination of the seating(s) of etioheme-1 in myoglobin substituted with a single bulky group at one of the four meso positions.

Implications for steric constraints to meso position access in heme degradation by coupled oxidation

Gerd N. La Mar, Jingtao Wang, Yiming Li, Dejian Ma, Heather Kalish, Alan L. Balch Depar tmen t o f Chemis try , Univers i ty o f Cali fornia, Davis, CA 95616 (e-mail: lamar@indigo , ucdavis , edu)

The highly stereoselective cleavage of hemin in myoglobin by coupled oxidation has been attributed to steric barriers that leave more space near the c~- than the other meso-positions. The steric barriers near meso positions in myoglobin have been investigated by establishing the thermodynamics and dynamics of possible seatings in the pocket of horse myoglobin of a four-fold symmetric etioheme I modified with bulky nitro and formyl groups at a single meso position. The cyanomet complex of these reconstituted myoglobins exhibits three sets of IH NMR resonances that are linked dynamically and exhibit intensity ratios that depend on the nature of the substitution. 2D 1H NMR has been used to assign the heroin and heine pocket resonances in the major isomer in solution and to determine tha! the heroin is oriented with the bulky meso group at the canonical )'-meso position of native hemin. The dominance of this isomer is attributed to the solvent exposure of this portion of the heroin which stabilizes the highly polar nitro and formyl groups. Using a combination of magnetization-transfer among methyl groups of the three isomers due to "hopping" of the hemin about its normal, the assigned resonances of an isoelectronic, bis-cyano complex of meso-nitro-etioheme I, and the known essentially constant rhombic perturbation of heme pocket sites on the hyperfme shifts of heme methyl, the two minor isomers are shown to place their bulky nitro group at the canonical 5-meso and ct-meso positions. The comparable population of the isomers with nitro or formyl groups at the hydrophobic ct- and 6-meso positions dictates that, while the static crystal structure finds more room near the ct-meso position, the deformation at minimal energetic expense near the ct- and 8-meso positions is comparable.

Neuroprotection against excitotoxic death by copper complexes of carnosine and its monofunctionalized cyclodextrin derivative

D. La Mendola a, F. Nicoletti b, E. Rizzarelli ~'c and G. Vecchio a a Dipar t imento di Sc ienze Chimiche, Universi ta di Catania, Viale A.Doria, 6 -95125- Catania (l taly) ;

Is t i tuto di Farmacolog ia , Scuola di Farmacia, Universi th di Catania, Viale A.Doria, 6 - 9 5 1 2 5 - Catania (I taly); c Is t i tuto per lo Studio del le Sos tanze Natura l i di In teresse A l imentare e Ch imico-Farmaceu t i co , CNR, Viale A .Dor i a ,6 -95125 -C a t an i a ( I ta ly) ,

Excitotoxicity revolves the destruction of neurons by glutamate and structurally related excitatory amino acids such as N-methyl-D-aspartate (NMDA) ~. Neurotoxicity mechanisms are complex. The presence of a large excess of calcium can be expected to activate many enzymes and to set in motion several destructive cascades. One of these has been shown to involve oxygen free radicals, superoxide and hydroxyl. For this reason, one possible pharmacological approach is to use molecules with antioxidant activities. Antioxidant activity is one possible function of the biological peptide camosine ([3-alanyl-L-histidine AH) which is present in millimolar concentrations in the skeletal muscle and brain tissue o f mammals z.

This study investigates the neuroprotection activities of carnosine, its mono cyclodextrin derivative [6-([3- alanylhistidine)-6-deoxy-13-cyclodextrin, CDAH], and their copper(II) complexes, on mixed cortical cultures containing both neurons and astrocytes prepared from:fetal mice. Experimental data show that ligands AH and CDAH alone have no protective effect in all ranges of concentrdt~on studied, while the copper(II) nitrate solution is shown to have an excitotoxic effect. The copper(II) comp'lexes of both AH and CDAH have a neuroprotective effect at 10 .4 M concentration and their efficacy is very similar. We can hypothesize SOD-like activity and hydroxyl scavenger action for both complexes.

1. D.W. Choi, J.Neurobiol., 23, 1261-1276 (1992). 2. A.A. Boldirev, A.M. Dupin, A.Y. Bunin, M.A. Babizhaev, S.E. Severin, Biochem. Int., 15, 1105-113 (1987).


An iron(II)-containing model system for catechol dioxygenases

Tung Suet Lain, Hung Wing Li, Thomas C. W. Mak, and Hung Kay Lee Department of Chemistry, The Chinese University of Hong Kong, HONG KONG (e-mail. O00410@cuhk. edu.hk)

Shatin, New Territories,

Catechol dioxygenases are non-heme iron enzymes which catalyze the oxidative cleavage of catechols by dioxygen, They are classified into the intradioLcleaving and the extradiol-cleaving dioxygenases. To date, a number of iron(III) complexes have been reported as models for the intradiol-cleaving enzymes. On the other hand, studies on iron(II)- catecholate model systems receive relatively less attention. Currently, we are interested in the reactivities of iron(II)- catecholate complexes toward dioxygen. Herein we report our recent progress in the development of an iron(II)- containing model system for catechol dioxygenases.

A mononuclear iron(II) armdo complex [(L)2Fe(TMEDA)] ( L = N(SiMe3)(2-CsH4N), TMEDA MezNCIq2CHzNMe 2 ) (1) has been prepared by the reaction of FeC12 with the lithium amide [LiL(TMEDA)]. X-Ray crystallography revealed that the iron center in 1 is coordinated by two bidentate amido ligands L and one chelating TMEDA molecule. Treatment of 1 with 3,5-di-tert-butylcatechol afforded the corresponding iron(II)-catecholate complex 2. The reactivity of 2 toward dioxygen has been studied by UV-Vis spectroscopy. Identification of the resulting degradation products is in progress.

1. Que, L., Jr., Ho, R. Y. N., Chem. Rev., 96, 2607-2624 (1996) 2. Pascaly, M., Nazikkol, C., Schweppe, F., Wiedemann, A., Zurlinden, K., Krebs, B., Z. Anorg. Allg. Chem., 626, 50-55 (2000)

This work was supported by a Direct Grant (A/C 2060127) of The Chinese University of Hong Kong.

Synthesis and characterization of mononuclear zinc complexes derived from monodentate and bidentate ligands

Tung Suet Lam, Hung Wing Li, Thomas C. W. Mak, and Hung Kay Lee Department of Chemistry, The Chinese University of Hong Kong, HONG KONG (e-mail: O00410@cuhk. edu.hk)

Shatin, New Territories.

The chemistry of zinc complexes has attracted much interest due to the important roles of this element in biological systems. Toward this end, a number of zinc complexes have been prepared as bionurnetic models for various zinc- containing enzymes. Encapsulating ligands, with tripodal ligands being the most popular ones, have been extensively used to support zinc complexes with coordination environments similar to those of the active site of the native enzymes. On the other hand, zinc compounds derived from monodentate and bidentate ligands receive relatively less attention.

A five-coordinate mononuclear zinc(II) arnidinate complex 1 has been prepared by the reaction of [LiL(TMEDA)] ( TMEDA = Me2NCH2CHzNMe2 ) with ZnC12. X-Ray crystallography revealed that the amidinate ligand L, the TMEDA and the chloride ligands constitute a distorted square pyramidal environment around the metal center. Current efforts to isolate the corresponding zinc(II) aryloxide complex by metathetical replacement of the chloride in 1 by an aryloxide ligand are underway in our laboratory.

1. Xu, X.-D., Lajmi, A. R., Canary, J. W., Chem. Commun., 2701-2702 (1998) 2. Chiou, S.-J., Innocent, J., Riordan, C. G., Inorg. Chem., 39, 4347-4353 (2000)

This work was supported by a Direct Grant (A/C 2060185) of The Chinese University of Hong Kong.


Labile dinuclear and tetranuclear manganese complexes of a phenolate-hinged dinucleating ligand

Frank Bartnik Larsen a, Astrid Boisen a, Alan Hazell b, Vickie McKee c, Christine J. McKenzie a Keith Murray e

Chemistry Institute, University of Southern Denmark, Odense Campus, Campusvej 55, 5230 Odense M, Denmark. (-email: f b l @ c h e m . s d u . d k ) ~Chemistry Institute, Aarhus University, Langelandsgade 140, 8000 Arhus. Denmark CChemistry Department, Loughborough University, Loughborough L E l l 3TU Leicestershire United Kingdom ':Department of Chemistry, Monash University, Clayton Campus, 3800, Victoria, Australia

Many proposals for the mechanisms of metal-containing enzymes and catalysts involve reactive intermediates incorporating water (or derivatives) as labile ligands. Intermediates with terminal hydroxide hgands with biologically relevant iron and manganese are notoriously difficult to isolate due to their reactivity. We present the structural characterisation of a mixed-valence dimanganese complex with two water ligands bound to the Mn(II) and two methoxide ligands bound to the Mn(III) in a H-bonded arrangement. This complex models plausible solvent interactions in dinuclear hydrolysis enzymes in particular the purple acid phosphatases. In the absence of alcohol a tetranuclear complex has been obtained. Preliminary results suggest the II/II/III/III oxidation state.

Structural dynamics of cytochrome c oxidase

Gisela Larsson a, Margareta Svensson-Ek a, So Iwata b, Peter Brzezinski a '~ Department of Biochemistry and Biophysics, Stockholm University, The Arrhenius Laboratories

for Natural Sciences, Stockholm University, 106 91 Stockholm, SWEDEN (e-mail: [email protected])

t, Department of Biochemistry and Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK

The membrane bound cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain in mitochondria as well as in many bacteria. It catalyses the reduction of molecular oxygen to water, and in the process creates a proton gradient across the membrane which is used to drive ATP synthesis. In spite of extensive research using a variety of techniques, the structure-function relation of COX is not fully understood. The structure of COX from Rhodobacter sphaeroides has been solved to a resolution of 2.3/2.8 A, as well as the structure of the inactive EQ(I-286) mutant ertzyme ~. E(I-286) is a key residue in the D-proton pathway, transferring protons both for pumping and for the reduction of oxygen at the active site. In the structure of the mutant enzyme, water molecules around the mutation site have been re-allocated as compared to the wild-type structure, and there is a shift of adjacent residues. We are now processing data from the ED(I-286) mutant enzyme, with a 50 % reduced pumping activity, which will add new information about the role of glutamate-286.

Our main focus is the structural dynamics of cytochrome c oxidase, combining spectroscopy and x-ray crystallography. We have designed a system to study the kinetics of COX in the crystals, enabling us to characterise the states of the crystals, and monitor transient intermediates. We aim to initiate the oxygen reaction with a laser-flash, and trap reaction intermediates by temperature control. If possible, the structures of these kinetic intermediates will be solved.

i. Svensson-Ek, M., Rodgers, L., Abramson, J., T6mroth, S., Brzezinski, P. and Iwata, S. (Submitted)


Dioxygen Binding to a Series o f Mononue lear Copper(I) Complexes with Linear Tridentate Chelators

David Lahti a, Karen Hatwell b, Susan Kaderli a, Kenneth D. Karlin b, Hong-Chang Liang b, Christiana Xin Zhang b, and Andreas D. Zuberbtihler a a Institute of Inorganic Chemistry, University of Basel, Spitalstrasse 51, 4056 Basel, SWITZERLAND

(e-mail: [email protected]) b Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA

1: R = H; X1 = X2 = N(CH3)2 The oxygenation of mononuclear copper(I) complexes R 2: R = Ell3; X 1 = X 2 = N(CH3) 2 with a series of linear tridentate chelators L (1 - 3) is i

xl CH2CH2--N--CH2CHz X2 3: R = H; Xl = N(GH3)2; X2 = 2-Pyridyl discussed and compared to the previously studied complex 4: R = CH3; X 1 = X 2 = 2-Pyridyl with bis[2-(2-pyridyl)ethyl]methylamine (MePY2, 4). In all

four cases, binuclear peroxo complexes Cu2L(O2) 2+ are formed with side-on binding of the 02 moiety, as found in hemocyanin. No direct evidence of superoxo species CuL(O2)* was obtained with any of the ligands, although such intermediates must be postulated for mechanistic reasons. Depending on ligand and solvent, various amounts of the isomeric bis ~t-oxo species Cu2L(O)22÷ are formed besides the peroxo complex. The interaction of CuL + with dioxygen can be described by:

2 CuL + + 02 :z~ C U 2 L ( 0 2 ) 2+ (3= 1); Cu2L(O2) z+ ~ decomposition products (2) Formation of Cu2L(O2) 1+ is significantly slower than with analogous binucleating ligands. With ligand 1 in acetone

the formation of the peroxo adduct becomes irreversible in the whole temperature range (183-273 K) studied, i.e. a back reaction (k_l) is not observed and an equilibrium constant cannot be obtained. This is in line with observations for other saturated (cyclic) ligands with an N3 donor set, but in contrast with the situation with open-chain ligands 2-4. Photochemical activation in the decomposition reaction (2) is observed for all systems. Zero or negative overall activation enthalpies for dioxgen binding (k~) are observed throughout (AH ~ = 0 to -27 k J/tool), reflecting the suggested composite nature of reaction (+1). In some cases, a change of solvent dramatically changes reactivity (e.g. for 4 in acetone or DCM. On the other hand the decomposition reaction (2) with 1 is superimposable for acetone and THF. In both solvents, photochemical activation predominates below 210 K, while thermal activation with AH ~ = 50 k J/tool and AS" = -40 J/(Kmol) is effective at higher temperatures.


Oxidative chemistry at dimanganese sites.

J e a n - M a r c L a t o u r , R 6 g i s Caspa r , L ione l D u b o i s

Department of Molecular and Structural Biology, CEA-Grenoble, 38054 Grenoble Cedex 9,France

Dimanganese sites exhibit a strong ability to react with peroxides either in natural processes such as in catalase enzymes where they disproportionate hydrogen peroxide or in industrial applications where they act as bleachnig agents. To get more insight into the mechanisms of action of the enzymes and catalysts we have developed a series of dimanganese complexes of a phenol-based hexadentate ligand (H-LBn) possessing a dangling benzyl group and studied their reactivity with hydrogen peroxide and m-chloroperbenzoic acid (mCPBA). The figure shows the X-ray structure of a prototype complex of the series [Mn2(OAc)2(L-Bn)(H20)](C104)2 where a terminal water molecule occupies a coordination site easily accessible to substrates.

These complexes present an intense activity toward hydrogen peroxide disproportionation and mechanistic studies clearly indicate that this activity involves shuttling between a p-oxodimanganese(II,III) and a bis-g-oxodimanganese(III,IV) species. The latter species has been identified by various spectroscopic techniques and prepared independently. The catalase-like activity can be suppressed in the

cr33) C145~ (3S CC4G)

C{;) C~4] 132~13 u

CI211CI ~) C{4 ;(441 C1421 0431

c{11) C{4:1

MN2I CV~

OSl) O{Sl)

cn

Figure : X-ray structure of the cation of [Mn2(OAc)2(L-Bn)(H20)](CIO4)2

presence of easily oxidizable substrates such as tritertiobutylphen01 which is oxidized into its radical. In the presence of mCPBA the complexes catalyze various oxygen transfer reactions.

Bioinspired oxidation of alkanes by bimetallic Fe(III)/M=O (M =Ru, Os) systems

Tai-Chu Lau, Shek-Man Yiu, Zhi-Biao Wu, Chi-Keung Mak Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, P. R. China

Methane monooxygenase (MMO) is one of the most remarkable enzymes in nature because it is able to catalyze the oxidation of the very inert methane to methanol. Spectroscopic and computational studies suggest that the active species that effects the oxidation of substrate is a di(p-oxo)diiron(IV) species. Inspired by MMO, we are interested to prepare bimetallic oxo species and study their reactivities toward the oxidation of organic substrates. We report here the stoichiometric and catalytic oxidation of alkanes by a FeC1jOsN(O)3 system. Although the OsN(O)3 ion is formally an osmium(VIII) species, it is a very weak oxidant that is indefinitely stable towards alkanes. Thus a solution of (Bu4N)[Os(O)sN] in CH2C12 containing cyclohexane (1 ml) remained unchanged for weeks at room temperature. However upon adding just a few equivalents of FeCI3 in acetic acid to the above mixture, cyelohexanol and cyclohexanone were produced within a few minutes at room temperature. It is proposed that the [Os(O)3N] coordinates to the Fe(III) center either through a nitrido or an oxo ligand (a double bridge is also possible) to produce a bimetallic species that is somewhat similar to the diiron active species in MMO. The oxidation can also be made catalytic by using ~BuOOH or H202 as the terminal oxidant.

The Research Grants Council of Hong Kong id acknowledged for financial support.


A comparison between native and recombinant forms of lactoperoxidase and myeloperoxidase by using organic and inorganic substrates as a probe

Enzo Laurenti a, Elena M. Ghibaudi a, Gianpaolo Suriano b'c, Nicole Moguilevsky b, Rosa Pia Ferrari a. ~' Dip Chimica I.F.M., Universitd di Torino, Via Giuria 7, 1-10125 Torino, Italy b Applied Genetics, Faculty of Science, UniversiM Libre de Bruxelles, ULB-IBMM, 12 Rue des Pr.

Jeener et Brachet, B-6041, Goseelies, Belgium Clnstitute of Molecular Pathology and Immunology, University of Porto, Rua R. Frias s/n, P-4200

Porto, Portugal

Lactoperoxidase (LPO) and myeloperoxidase (MPO) are involved in an anti-bacterial defense system, based on the peroxide-mediated oxidation of halides and pseudo-halides to give bacteriostatic and bactericidal agents (like C10- and SCNO) I.

In this study, we compared the structural and functional properties of different engineered forms of LPO and MPO 2, by using four potential substrates as a probe: SCN, catechol and two of its analogues, dopamine and 3,4- dihydroxyphenylpropionic acid (DHPP). The following enzymatic forms have been investigated: native bovine LPO, recombinant wtMPO, recombinant bovine and human LPO and three mutants of bovine LPO.

The enzyme-substrate interaction has been studied by UV-Vis and EPR spectroscopy. Binding studies and kinetic measurements results have been interpreted in the light of the MPO X-ray structure 3 and of the LPO structural model 4.

1. Dunford H.B., Heme Peroxidases (1999) Wiley-WCH 2. Kooter I.M., Moguilevsky N., Bollen A., Sijtsema N.M., Otto C., Dekker H.L. and Wever R., Eur. J. Biochem., 274,

26794-26802 (1999) 3. Zeng J. and Fenna R.E., J. Mol. Biol., 226, 185-207 (1992) 4. De Gioia L., Ghibaudi E.M., Laurenti E., Salmona M. and Ferrari R.P., JBIC, 1,476-485 (1996)

Low-Frequency Fourier Transform Infrared (FTIR) Spectroscopic Studies Related to the Oxygen Evolving Complex of PSII

Neil A. Law a, Warwick Hillier a, Hsiu-An Chu a, Wen-Yuan Hsieh b, Shannon Haymond a, Jose F. Cerda a, Vincent L. Pecoraro b, Gerald T. Babcockao

Department of Chemistry, Michigan State University, East Lansing, MI 48824-1322 USA b Department of Chemistry, University of Michigan, Ann Arbor, MI48109-1055 USA (email: [email protected])

The Oxygen Evolving Complex (OEC) of Photosystem lI (PSII) catalyzes photosynthetic water oxidation. The OEC requires a tetranuclear cluster of Mn ions, at least one chloride ion, a calcium ion, and specific amino acid residues, for example Tyrosine Z (Yz). Vibrational spectroscopy offers one means to probe the structure and intricate mechanism of water oxidation, which are yet to be fully understood. Using difference Fourier transform infrared spectroscopy (FTIR) we have recorded spectra for PSII in the mid-IR (1800-1000 cm-1) and low-frequency (<1000 cm -I) region of the IR spectrumJ The low-frequency region, however, offers the potential to explore the metal-ligand interactions critical to the water oxidation mechanism. Therefore, we have developed a methodology for recording difference FTIR spectra in this IR regionJ Recently, through difference FTIR, we have reported evidence for an OEC vibrational mode that is consistent with either a [Mn0a-O)]2 or a [Mnz0.t-O)] motif at 606 cm l. 1,20xo-bridged Mn structures are generally proposed for the OEC. l Vibrational mode signatures for [Mn(p-O)]z cores have been identified for model compounds in the 600-700 cm -~ 1R region, however, data assigning these vibrational modes are scant, l Therefore, we have undertaken a model compound study of [Mn(~-O)]2 complexes by resonance Raman and FTIR, in order to interpret the OEC data more completely. Results from that study, and from our ongoing probe of low-frequency FTIR PSII sample preparations will be described.

1.Chu, H.-A.; Hillier, W.; Law, N. A. & Babcock, G.T. Biochim. Biophys. Acta, 1503, 69-82 (2001) 2.Chu, H.-A.; Sackett, H. & Babcock, G. T. Biochemistry, 39, 14371 -14376 (2000)

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