abstracts poster presentations (p)

Journal of Inorganic Biochemistry 86 (2001) 271

New insight into the roles of the structural Ca 2+ ions of horseradish peroxidase

Barry_ D. H o w e s a, A les sandro Feis a, Laura Ra imond i b, Chiara Indiani a, and Giul ie t ta Smulev ich a,

" Dipartimento di Chimica, Universit5 di Firenze, ITALY (e-mail: [email protected], it) b Dipartimento di Farmacologia Preclinica e Clinica, Universit5 di Firenze, ITALY

Electronic absorption and resonance Raman spectroscopy are used to investigate the extent to which the heme pocket architecture of horseradish peroxidase isoenzyme C (HRPC) is determined by the two structural C a 2+ ions. Ca-depletion, by removal of o n e C a 2+ ion, results in a modification of the Fe(III) heme states, with an uncommon quantum mechanically mixed-spin state becoming the dominant species. The spectra of complexes formed with fluoride, benzohydroxamic acid and CO demonstrate that changes to the distal cavity structural properties upon Ca-depletion are easily reversed. In contrast to the native protein, the Ca-depleted ferrous form displays a low-spin bis-histidyl heme state with a small proportion of high-spin heme also evident. The v(Fe-Im) stretching mode downshifts 27 cm 1 upon Ca- depletion revealing a marked structural alteration of the proximal cavity near the histidine ligand. The specific activity of the Ca-depleted enzyme is 50 % that of the native form. The effects on enzyme activity and spectral features resulting from Ca-depletion are reversible upon reconstitution of the enzyme. Analysis of the present results together with the overall body of data available for HRPC suggests that the proximal Ca 2÷ ion is lost.

The EU (contract BIO4-97-2031) and the Italian Ministero dell'Universita' e Ricerca Scientifica e Tecnologica (MURST) are acknowledged for financial support.

Ruthenium-manganese complexes for solar energy conversion

P H u a n g Kendz a, A M a g n u s o n Styr ing a, R L o m o t h b, A Tran ¢, J Fryxel ius c, H Schmidt , H Berg lund

Baud in b, M A b r a h a m s s o n b, J H 6 g b l o m a, J Park a, B van Rot te rdam, B A k e r m a r k c, L H a m m a r s t r 6 m b,

L Sun c, S Styr ing a

"Department of Biochemistry, Lund University, P.O.Box, 124, S-22100 Lund, Swden (e-mail: [email protected]); bDepartment of Physical Chemistry, Uppsala University, Box 532, SE-75121 Uppsala, Sweden;CDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden

We present progress in the development of artificial photosynthesis, as a means to harvesting and storage of solar energy. Our goal is to utilize water as sacrificial electron donor, which will allow a net production of reducing equivalents, and the ultimate production of fuel. The only light-driven molecular catalyst for water oxidation exists in Photosystem II (PSII), which has a tetranuclear Mn-cluster in the active site. Here we present several Mn-compounds, that we have developed for the purpose of creating water-oxidizing catalysts.

Ru-tris(bipyridine) derivatives, which mimic the function of the primary donor in PS II, were linked with manganese containing moiety, mimicking the Mn-cluster on the PSII donor side. We have constructed a number of heteronuclear complexes, containing a Ru-photosensitizer and various Mn-complexes I. The compounds have been characterized with regards to their photophysical and photochemical properties, redox potentials and structure. The most promising compounds are capable of undergoing several electron transfers from the Mn-complex to the photosensitizer, leaving 3 to 4 oxidizing equivalents on the Mn. In the latest development, we have constructed ligands that stabilize higher oxidation states in Mn, in order to promote formation of MnV=O species, which is believed to be an intermediate in the water oxidation mechanism.

1. Sun L., Hammarstr6m L., Akermark, B. and Sty-ring, Stenbj6rn., Chem. Soc. Rev., 30, 36-49 (2001)

Knut and Alice Wallenberg Foundation, The Swedish National Energy Administration, DESS, NFR, TFR and TMR (CT96-0031) are acknowledged for financial support.

272 Journal of lnorganic Biochemistry 86 (2001)

Copper delivery from the metallochaperone HAH1 to the Wilsons disease protein

David L. Huffman a, Jenny Kwak a, Thomas V. O'Halloran ~'b a Department o f Chemistry and b Department o f Biochemistry, Molecular Biology, and Cell

Biology, Northwestern University, 2145 Sheridan Road, 60208, Evanston, Illinois, USA, (e-mail: d-h uffman @norwestern. edu)

The Wilsons disease protein (ATP7B) is a P-type ATPase responsible for copper trafficking across vesicular membranes. Mutations of ATP7B lead to deleterious copper accumulation, notably in the brain, liver, and kidneys. ATP7B possesses six N-terminal MTCXXC metal-binding motifs within a [3cx[313et[3 structural domain, by homology to solution structures in related P-type ATPases. ~.2 We have synthesized the gene for the fifth metal-binding domain of the Wilson disease protein (WND5) and the gene for the human metallochaperone HAH1. HAH1 and WND5 were then overexpressed and purified to homogeneity. The Cu(I)-loaded forms of these proteins were prepared, followed by incubation with partner apoprotein. We demonstrate facile and reversible copper delivery to partner, regardless of the initial donor, with an equilibrium constant for copper exchange of 15+1 (Figure). Mechanistically, we propose that a low activation barrier for copper transfer facilitates rapid transfer between chaperone and parmer. 3 Homology modeling of the Wilsons metal-binding domains using Menkes domain 41 as a guide reveals conserved features that are likely important for partner recognition.

I. Gitschier J., Moffat B., Reilly D., Wood W. I. and Fairbrother W. J., Nat. Struct. Biol., 5, 47 - 54 (1998) 2. Banci L., Bertini I., Ciofi-Baffoni S., Huffman D. L. and O'Halloran T. V., J. Biol. Chem., 276, 8415-8426 (2001) 3. Huffman D. L. and O'Halloran T. V., J. Biol. Chem., 275, 18611-18614 (2000)

NIH (TVO), NIH-Postdoctoral Grant (DLH), and the Robert H. Lurie Comprehensive Cancer Center

Studies on new tumour-active metal complexes

Fazlui Huq b, Philip Beale a, Jun Qing Y u b

Royal Prince Alfred Hospital, Gloucester House, Missenden Road, Camperdown, NSW 2050, Australia)

b School o f Biomedical Sciences C42, University o f Sydney, East Street, Lidcombe, NSW 2141,Australia (e-mail: f [email protected], edu.au)

The reason why transplatin is toxic rather than being anticancer active is believed to be associated with its higher reactivity than cisplatin. It is therefore thought that the introduction of sterically hindered planar ligands may reduce the reactivity of the complexes sufficiently so as to result into tumour-active compounds with a different spectrum of activity.

We have prepared a number of transplanaramine platinum(II) of the type trans-Pt(II)L2C12 and trans-Pt(II)L(NH3)C12 where L stands for a planar heterocyclic ligand. Some of the complexes have shown activity against a number of ovarian and other cancer celMines. This poster describes the activities of the designed complexes and the nature of their interaction with DNA.

Journal of lnorganic Biochemistry 86 (2001) 273

Modelling the interaction between Cu 2+ and the nucleobases adenine and guanine in solution in water

Fazlul Huq and Farhad Mohammad " School of Biomedical Sciences C42, University of Sydney, East Street, Lidcombe, NSW2141,

Australia

The interaction between metal ions and DNA and its constituents has been the subject of many different studies. However, in many cases there is controversy on the exact nature of the binding between metal ions and nucleobases, nucleosides, nucleotides and DNA, especially applying to solutions. We have followed the interaction between Cu 2+ and nucleobases adenine and guanine in solution in water by UV-visible spectrophotometry using the technique of continuous variation combined with molecular modelling. The maxima in the UV difference spectra suggest that copper(II) forms 1:1, 1:2 and 1:3 adducts with adenine, and 1:1 and 1:2 adducts with guanine. The proposed structures have been optimised based on molecular mechanics and semi-empirical calculations

Characterisation of structural, dynamic and electronic properties of metal centers in proteins by high-resolution EPR techniques: iron sulfur proteins

Jfirgen Hiittermann and Reinhard Kappl Institut f~r Biophysik, Klinikum, Universit~it des Saarlandes, 66421 Homburg, FRG

Iron sulfur centers accomplish versatile tasks in biological systems ranging from catalysis to electron transfer. High potential iron-sulfur proteins (HiPIPs) are involved in electron transfer in bacterial photosynthesis and have attracted considerable interest because of the high redox potentials. In the oxidized paramagnetic state the [4Fe4S] 3+ cluster contains a delocalized mixed-valence iron pair with formal charge +2.5 and two ferric irons coupled antiferromagnetically to an effective spin S=1/2. We have applied orientation selective cw- and pulsed-ENDOR spectroscopy to frozen solutions of several HiPIPs of different origin, including mutants. The distribution of spin population, the valence states of the irons and the orientation of the g-tensor with respect to the cluster geometry could be derived. The obtained g-tensor orientations as well as the assignment of the two iron pairs can be correlated to the different cluster species appearing in EPR spectra of oxidized proteins. The findings are compared to results from model structures, to NMR-data and to X-ray-structures of the proteins.

In 2Fe2S-proteins, the paramagnetic reduced [2Fe2S] + cluster comprises a ferric and a ferrous iron with localized valencies, which are antiferromagnetically coupled to a S=l/2-system. The analysis of the interactions of amino acid protons in the vicinity of the cluster of Arthrospira platensis allowed to identify the valence state of each iron and the orientation of the g-tensor within the cluster. It turned out that the tetrahedral coordination of ferrous site is dominantly determining its alignment.

The knowledge of the orientation of magnetic axes in such types of clusters is important fbr the interpretation of magnetic interactions observed by EPR in multi-center systems like molybdenum containing hydroxylases and for the arrangement of individual units in inter-molecular interactions. Futhermore the sensitivity of magnetic properties may allow to conclude on determinants of the redox potential. Finally, spectral properties of EPR- and ENDOR-resonances can be exploited to obtain a correlation to intrinsic disorder and flexibility of protein residues around the clusters.

274 Journal of Inorganic Biochemistry 86 (2001)

3 th Generat ion ironporphyrins as b iomimmet ic oxidation catalyst

Yassuko Iamamoto a, Aria Paula Jecks Maestrin a, Andr6a Junqueira de Melo Maurin b and Pierina Sueli Bonato b. " Depar tamento de Quimica, F F C L R P - U S P Avenida Bandeirantes , 3900, CEP: 14040-901 Ribeirdo

Preto, Sdo Paulo, Bazil , Fax: 55-16-6338151, e-mail: i amamoto@usp .br Depar tmento de Fis ica e Quimica, F C F R P - U S P , Avenida do Cafk,s/n, Ribeir~o Preto, S~o Paulo, Bazil

The oxidation of substrates such as cyclohexene with polychlorinated porphyrins as FeTNPClsPC1 (1) as catalyst lead to the formation of 80% of epoxycyclohexane based on iodosylbenzene as oxidant, however there is simultaneous formation of allylic alcohol and ketones with apparent total yields > 100% indicating that radical processes are involved I. When FeT2TFMPC18PC1 (2) was used we observed only epoxides as product. The biomimetic behavior of this catalyst is due to the 2-CF3-phenyl groups which are responsible for hydrophobic active site. This behavior was verified with drugs oxidation studies, for example in the oxidation of propafenone, only 2 lead to the active metabolite, 5-hidroxypropaphenone. It is evident the central role of radical n-cation, (Fe(IV)OP'+), as active species explaining the very good conversions to the epoxides as expected for cytochrome P-450 models 2. We believe that 2-CF3- groups, a less electronegative group in 1, if compared to NO2- groups in 2 accomplish an important role preventing the triggering of radical chain processes.

m= 4

6

Porphyrin R

1 FeTNPC18P 2-NO2-Phenyl C1 2 FeT2TFMPC18P 2-CF3-

Phenyl C1

l .

2. M.D. Assis and J.R. Lindsay Smith, J. Chem. Soc., Perkin Trans. 2 (1998) 2221. J.T. Groves and Y.Z. Han, in "Cytochrome P-450: Structure, Mechanism, and Biochemistry ", (P.R. Ortiz De Montellano, ed.) Plenun Press, New York, 1995, Chap. 1. Acknowledgment: FAPESP, CAPES, CNPq

Synthesis and characterization of Co(II), Ni(II) and Cu(II) complexes with therapeutic l igands

Nicoleta Iftimie a, Greta Mihaela Patrinoiu b, Luminita Patron b, N. Stanicab and Aurelia Meghea a a Univers i ty "Pol i tehnica '" o f Buchares t , Facul ty o f Indus t r ia l Chemistry, 1 Pol izu Street , 78126, Bucharest , ROMANIA, (e-mail: [email protected] . ro ) b Ins t i tu te o f Phys i ca l - Chemis try "I. G. Murgu le scu" , Coordinat ion Chemis try Laboratory , Splaiul Indepensen te i no. 202, 70208 Buchares t , ROMANIA; e-mail: f t u n a @ p e n e t . p c n e t . r o

Interest in metal complexes of biguanide derivatives arises from their well acclaimed medicinal values as germicidal, bacteriostatic, hypoglycaemic and anticarcinogenic agents.

Also, biguanide and biguanide derivatives are extremely powerful and formidable coordinating ligands which have played important roles in elucidating many interesting aspects of coordination chemistry.

We have initiated studies on the coordination chemistry of the antiviral drug of

biguanidetransition metalfamilYions.- morpholinebiguanide hydrochloride (Fig.) - with some JJI~--NH CI[ NH2"HCI

The synthesis, spectroscopic and magnetic properties are reported for N H N H coordination complexes of Co(II), Ni(II) and Cu(II) with the antiviral drug morpholinebiguanide hydrochloride (MBig) known as "Moroxydine" and some biologically important aliphatic carboxylic acids (oxalic, malonic, tartaric). In all studied complexes, the morpholinebiguanide drug acts as a bidentate Iigand through nitrogen atoms of the =NH groups.

Pro or antioxidant effects of these compounds were tested by chemiluminescence experiments, and have been correlated with their therapeutic activity.


Transient protein complexes studied by paramagnetic NMR: the complex of nitrite reductase and pseudoazurin

A.Impagliazzo, M. Ph. Verbeet, G.W. Canters, M. Ubbink Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands, (e-mail: [email protected], nl)

Nitrite reductase (110 kDa) and pseudoazurin (14 kDa), both copper proteins, form a transient protein-protein complex essential in the denitrifying pathway in the bacterium Alcaligenesfaecalis S-6. The complex between NIR and pseudoazurin is formed in order to enable the electron transfer from pseudoazurin to NIR and then to nitrite. It is unknown how pseudoazurin binds to NIR. We have labelled the pseudoazurin with 15N and 13C and assigned the backbone of the protein. We have studied the complex with NMR in two oxidation states (ox-ox and red-red). In both cases, in the HSQC spectra the resonances of pseudoazurin do not shift upon complex formation. The intensity decreases with the fraction of bound protein and some line broadening is observed, suggesting a slow exchange between the two proteins at the NMR time scale. The dissociation rate constant was calculated to be -20 s -~ and the binding constant - 4 xl 05 M -1. We have produced perdeuterated protein in order to obtain information of pseudoazurin in the bound form. The final aim of this project is to determine the structure of the complex using paramagnetic NMR methods.

The efficient hydrolysis reaction of phosphate ester ligated to zinc(II) complex promoted by general acid.

Masami Ito, Koyu Fujita Research and development Center, Oita University, Dannoharu 700, 870-1192, Oita, Japan, (e-mail. [email protected]).

I-2 Hydrolytic enzymes such as phosphatases or nucleases are known to catalyze the hydrolysis of phosphate ester.

Many of these enzymes contain metals such as zinc at the active sites. The catalytic role of zinc is ascribed to the binding and activation of substrates, while deprotonation of coordinated water to produce a nucleophilic zinc hydroxide is also proposed as an essential catalytic hydrolysis function. It is well known that model complex system can mimic this

z enzymatic function under basic condition. However, one of these enzymes, such as P1 nuclease have relatively low optimal pH condition (pH 4-6) for their catalytic reaction. From inorganic point of view, it seems unlikely that the zinc(II)-bound water can deprotonate to form the hydroxo moiety at such low pH condition. Thus, nucleophilic zinc hydroxide may not be involved in their catalytic reaction. The possibility of other reaction mechanism can not be neglected at the present stage.

In this report, we will describe the hydrolysis reaction of ligated phosphate complex in Zn II phosphate complex,

[(TPA)ZnII(BNP)](C104) (2) (TPA = Tris(2-pyridylmethyl)amine BNP = bis(p-nitro-phenyl) phosphate) which was

structurally characterized. It was confirmed by potentiometric titration and ESI-MS that the ligation of BNP to Zn(II) complex in aqueous solution was dominant at every measured pH condition. The pH-hydrolysis rate profile suggested that the hydrolysis reaction of phosphate ester in 2 was promoted under acidic aqueous media more efficient than the alkaline aqueous media. Thus, lewis acidity of zinc(II) ion promotes the hydrolysis reaction of phosphate ester efficiently under acidic condition. The relevancy of this system to P1 nuclease which has low optimal pH condition is also disscused.

1. Lipscomb N.W. and Strfiter N. Chem. Rev. 96, 2375-2433, (1996)and references therein. 2. Wilcox D.E., Chem. Rev. 96, 2435-2458(1996).


Catalytic intermediates in heme catalases and peroxidases: multifrequency (high-field) EPR investigation of tyrosyl radicals.

Anabella Ivancich, Sun Un Section de Bio~nerg~tique, Departement de Biologie Cellulaire et Moleculaire. URA CNRS 2096.

CEA-Saclay. (e-mail. ivancich@dsvidf, cea.fr)

Protein-based radicals are involved in the redox chemistry of metalloproteins I. tryptophanyl radicals have been proposed to have specific functional roles in electron and/or proton transfer process in several enzymes. A heme iron constitutes the catalytic site of peroxidases and catalases, the enzymes that are responsible for the regulation of the cell concentration of hydrogen peroxide, a reactive oxygen species. Our studies on the catalytic cycle of different catalases and peroxidases, show that the formation of the [Fe(IV)=O Tyr °] intermediate, as the result of intramolecular electron transfer between the porphyrin and a tyrosine residue, is a common strategy to both enzymes. The protein environment of the Tyr °, which we probe by measuring the accurate g-values obtained by high-field EPR 2, is conserved for the different enzyme intermediates whether the site for the radical formation is conserved or not. A pH-

In particular, tyrosyl (Tyr') and

heme

~'~\,~. His~ 61

Asp334 .~, J

• water -

dependent formation of the [Fe(IV)=O por "+] or the [Fe(IV)=O Tyr'] intermediate was observed, in particular in plant peroxidase isozymes. A relation between the redox potential and the nature of the radical intermediate (por "~ or Tyr') is proposed 3

1. Stubbe, J.; van der Donk, W. A. Chem. Rev., 98, 705-762 (1997). 2. Ivancich, A.; Mattioli, T. A.; Un, S. J. Amer. Chem. Soc., 121, 5743-5753 (1999). 3. Ivancich, A.; Mazza, G.; Desbois, A., Biochemistry, in press (2001).

Reagentless biosensors based on Ru-complex modified carbon-paste/enzyme electrodes

E.V. I v a n o v a a, V.S. S e r g e e v a a, A . D . R y a b o v a, W. S c h u h m a n n b

Department of Chemistry, Moscow State University, 119899, Chemical department, Chair of chemical enzymology, Moscow, Russia, (e-mail: ivanka@enzyme, chem. msu. ru) b Ruhr-Universitiit Bochum, Anal. Chem. - Elektroanalytik& Sensorik, D-44780 Bochum, Germany

The function of amperometric biosensors is related to the electron-transfer process between the active site of an (immobilized) enzyme and an electrode surface. Direct electron transfer is often difficult since the active site of the enzyme is deeply buried within the protein shell. An altemative electron-transfer pathway is based on free- diffusing low molecular weight redox active molecules.

A new class of such redox mediators with the common formula Ru(LL)2(X)2. where LL=l,10-phenantroline or 2,2'-bipyridine type ligands, and X is an acido ligand) have been synthesized. The attractive features of these mediators are theft broad activity spectra with respect to their interaction with different types of dehydrogenases.

Two approaches for the development of related biosensors have been realized. In the first, the enzyme had been integrated in a carbon paste already containing the redox mediator, and in the second the enzymes were immobilied on an mediator-containing carbon-paste surface using a Nation film.

The typical chronoamperometric response of such a biosensor is presented in the figure. The electrodes with incorporated enzyme showed an extend linear measuring range for the substrate

t,me ¢ ~ ¢

Chronoamperometric response for CPE, modified with

Ru(phen)2C12. covered with NAD+-dependent glucose dehydrogenase entrapped

within a Nation film


Bioinspired Iron(II/III) Complexes as Catalysts and Molecule-Based Magnets

Erns t -G . J~iger, G u i d o L e i b e l i n g , M a r c R u d o l p h , H e l m a r G6r l s .

Department of Chemistry, University of Jena, August-Bebel-Strasse 2, 07743 Jena, Germany, (e-mail: [email protected])

Iron(II/III) complexes of specially substituted macrocyclic IN4 2-] coordinated or open-chain [N2022-] coordinated Schiff base ligands are suitable to mimic the structure (distances, angles) and the spin state (low spin, intermediate spin, high spin) of many active sites of heme enzymes, l With tridentate [NO2 2] ligands, several very stable dinuclear O,O-bridged iron(II) complexes with a broad variety of additional ligands and coordination numbers can be isolated. The catalytic performance of such complexes with respect to the activation of dioxygen is strongly determined by peripheric substituents that are able to form intra- or intermolecular H-bridges. Intermolecular H-bridges in supramolecular structures are also responsible for cooperative magnetic properties such as ferromagnetic behavior at low temperature or the very sharp and strongly reversible low-spin-/high-spin transition at 328 K with a significant hysteresis and a AT of only 4 K in the bis(imidazole)adduct an Figure 1.2 The corresponding high-spin mono-adduct with imidazole resembles the core of oxgen-free myoglobin.

rQ

Figure 1

1. J~iger, E.-G.., in Chemistry at the Beginning of the Third Millennium, (L. Fabbrizzi, A. Poggi, Eds.), Springer-Verlag Berlin Heidelberg New York, 103-138, 2000. 2. Mtiller, B.; Leibeling, G.; J~iger, E.-G.; Chem. Phys. Letters 319, 368-374, 2000. Acknowledgment: The authors thank the Deutsche Forschungsgemeinschaft, the Freistaat Thtiringen and the Fonds der Chemischen Industrie for financial support.

Molecular engineering of catalase-peroxidases

Christa Jakopitsch a, Regelsberger Gtinther", Florian Riiker b, Gfinter A. Peschek b, Christian Obinger a ~Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-I190 Vienna, Austria, Fax. +43-1-36006-6059; Email: [email protected] blnstitute of Applied Microbiology, University of AgriculturaI Sciences, Muthgase 18, A-1190 Vienna Clnstitute of Physical Chemistry, University of Vienna, Althanstrafle 14, A-1090 Vienna

Class I of Peroxidase Superfamily I (enzymes from plants, fungi and bacteria) contains cytochrome c peroxidase (CCP), ascorbate peroxidases (APX) and catalase-peroxidases (KatGs) with high homology at the active site. In all Class-I peroxidases, the proximal histidine has an imidazolate character because its 8-nitrogen is H-bonded to a conserved aspartate residue which is also H-bonded to a conserved tryptophan residue. The high-resolution crystal structures of both CCP and APX indicate the presence of the triad Arg-Trp-His at the distal side as well as a H-bond from the 8-nitrogen of distal His to a near surface asparagine. Sequence alignments suggest the presence of these residues also in the distal pocket of catalase-peroxidases. Both CCP and APX do not exhibit substantial catalase activity. On the contrary, bifimctional KatGs are the only members of Superfamily I with a catalase activity comparable to monofunctional catalases, but the catalase activity of KatGs displays several unique distinguishing characteristics. Additionally, KatGs function as broad-specificity peroxidases. Here, we present a comprehensive mutational, spectroscopic and kinetic study on catalase-peroxidase from the cyanobacterium Synechocystis PCC6803. Recombinant proteins with mutations at distal Arg, Trp and His as well as at proximal Asp, His and Trp were investigated by both steady-state and transient-state spectroscopy. The role of these active site residues in the bifunctional (catalase versus peroxidase) activity of KatGs is discussed in comparison with data known from both APX and CCP.


Oxovanadium(IV) and copper(II) complexes of reduced Schiff-bases

Tamfis Jakusch a, I sabe l Cor re ia b, JoSo Cos ta Pessoa b, Tam,is Kiss a

Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440, 6701 Szeged, HUNGARY (e-mail: jakusch@chem, u-szeged, h u)

b Centro Quimica Estrutural, Instituto Superior TOchnico, Avenida Rovisco Pais, 1096 Lisboa, P OR TUGA L

A crucial point of VO(IV) and Cu(II) binding with proteins, peptides and their derivatives is the metal ion induced deprotonation and co-ordination of the amide-nitrogen. In the present work the interaction of the Schiff-base derivatives SalH2GG and SalH2GGG was studied with VO(IV) and Cu(II) by combined pH-metry and EPR and UV-VIS spectroscopy. The ligands were prepared by the reduction of the Schiff-bases derived form salicylaldehide and GG or GGG.

The SalHzGG forms 1:1 complexes with both metal ions. In the ML type species the fenolate and the amino group co-ordinate to the metal ions. At pH 5-6, MLH.~ is formed with a 6+5+5 joint chelate system, through the deprotonation of the amide-nitrogen. At pH > 7 the VO(IV) complex deprotonates and forms a temary hydroxo species, while CuLH_~ does not hydrolyse even at pH 11.5.

The Cu(II) induces deprotonation of the two amide-nitrogens in SalH2GGG, the first occurs at pH -5-6, while the second at pH-9. This latter pK value is very similar to that of the last amide pK of tetragly, when the amide-nitrogen occupies the forth equatorial position. The VO(IV) is not able to induce the amide-nitrogen deprotonation in the acidic and neutral pH range, and a bis complex (II) predominates at pH > 4.5. The amide deprotonation rises just above pH 9, parallel with the hydrolysis of VO(IV).

I

II

The authors thank the project OTKA T31896, FEDER and FCT (POCTI/35368/QUI/2000) for fmancial support and the Hungarian-Portuguese Intergov. S&T Coop. Program for travelling funds.

Mechanistic studies of the activation of isoniazid by M. tuberculosis catalase-peroxidase and manganese

B. J a m a r t - G r r g o i r e a, J. Bodigue l , a J. Nagy , b N. Brosse , a K. A. B r o w n b

'~Laboratoire chimie organique MAEM UMR mixte CNRS-UHP n ° 7567, FacultO des Sciences, UniversitO H. PoincarO Nancy 1, Bid. des Aiguillettes BP 239 F-54506 Vandoeuvre-lks-Nancy France ( e-mail: [email protected]) bDepartment of Biology and Biochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AY, United Kingdom

Isoniazid (INH), an antibiotic used in the treatment of tuberculosis, is a prodrug requiring activation by the catalase- peroxidase (CP) from Mycobacterium tuberculosis (Mt). The active form of INH and activation mechanism are not conclusively known. In an effort to better define the reaction pathways leading to INH activation, we report the synthesis of the two singly ~SN-labeled isotopic isomers of INH and the studies of their oxidation by CP from Mt andJor manganese. The two singly 15N-labeled isotopic isomers of INH were obtained in 3 and 5 steps starting from 15N potassium phthalimide.~ HPLC studies demonstrated that Mn don't play an essential part in the INH oxidation by CP from Mt. More importantly, mass spectrometry analyses of reaction products obtained using singly labeled 15N-isoniazid isotopic isomers suggests that formation of the isonicotinamide reaction product involves splitting of the C-N bond of isoniazid, rather than the N-N bond as previously proposed. The reaction mechanism proposed here thus includes the formation of an isonicotinic acyl radical intermediate. 2

1. Jamart-Grrgoire B., Brosse N., Bodiguel J., Synthesis, 269-270 (1998) Brosse N., Pinto M. F. and Jamart-Grrgoire B., J. Chem. Soc., Perkin Trans. 1, 3685-3688 (1998) 2. Bodiguel J., Nagy J.M., Brown K.A., Jamart-Gr6goire B., J. Am. Chem. Soc., 123, 3832-3833 (2001).

The commission of european communities is acknowledged for support


Role of terminal and side chain donor groups in the dimethyltin(IV) induced amide deprotonation

Attila Jancs6 a, Tamfis Gajda a, Bernard Henry b, Patrice Rubini b aDepartment o f Inorganic and Analytical Chemistry, University o f Szeged, D6m t~r 7. Szeged, H-

6720, HUNGARY (e-mail: [email protected], hu) bLaboratoire de Chimie Physique Organique et Colloidale, UMR SRSMC CNRS 1l ° 7565,

Universit~ Henri Poincar~- Nancy 1., B.P. 239, F-54506, Vandoeuvre-lbs-Nancy Cedex, France

Organotin cations are known to have biological and pharmaceutical relevance thus they have been extensively studied in terms of their coordination chemical behaviour towards different bioligands. These studies can provide information on the bioavailability and possible effects of such metal ions on biochemical processes. As a continuation of our previous studies on different dipeptides I we investigated the equilibrium and solution structure of the o dimethyltin(IV) complexes of two other dipeptides (Gly-Asp and Asp-Gly) 2 and a peptide-type ligand (2-hydroxyhippuric acid) 3. The pH-metric and IH/13C NMR experiments proved the . c. "9 / \crt2 formation of slow ligand-exchanging complexes in the neutral pH-range with {COO , N-amide, ~136°H33 *(~S! *ZZ-'N'/ NH2/O-phenolate} coordination in a trigonal bypiramidal manner. When the amide group is c" ~ c = o deprotonated by dimethyltin(IV) anchored at the carboxylate group, the binding strength of the ligand is governed by the electrostatic Sn--OOC and the covalent Sn-N- bond, while the nature of the other terminal chelating donor is less determining.

1. Surdy P., Rubini P., Buzas N., Henry B., Pellerito L., Gajda T., Inorg. Chem. 38, 346-352 (1999) 2. Jancs6 A., Henry B., Rubini P., Vank6 Gy., Gajda T., J. Chem. Soc. Dalton Trans., 1941-1947 (2000) 3. Jancs6 A., Gajda T., Szorcsik A., Kiss T., Vank6 Gy., Henry B., Rubini P., J. Inorg. Biochem., 83, 187-192 (2001)

The fmancial support of the Hungarian Research Foundation (Project No.: OTKA T025114) is gratefully acknowledged.

A comparative study of horseradish peroxidase, lactoperoxidase and myeloperoxidase compounds I and II reactivity towards indole derivatives

Walter Jantschko a, Mar io Al legra b, Paul Georg FurtmiJller a, Christian Obinger a

"Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria, Fax. +43-1-36006-6059; (e-mail. [email protected]) ~Department o f Pharmaceutical Toxicological and Biological Chemistry, Univervity of Palermo, Via Carlo Forlanini, Palermo, Italy

The indole moiety is present in a great number of biologically important molecules, e.g. the plant growthhormone indoleacetic acid, the pineal gland hormone melatonin, serotonin and tryptophan. Here, we focus on the oxidation of the indole derivatives tryptamine, N-acetyltryptamine, tryptophan, melatonin and serotonin by the peroxidase intermediates compound I and compound II. The reactions with enzymes from both peroxidase superfamilies have been investigated by the sequential-mixing stopped-flow technique. Bimolecular rate constants of the redox intermediates of horseradish peroxidase (superfamily I of enzymes from plants, fungi and bacteria) and lactoperoxidase (LPO) and myeloperoxidase (MPO) -both from superfamily II of animal and human peroxidases- are presented (pH 7 and 25°C) and discussed with respect to known differences in the heme pocket of these proteins. Principally, serotonin (5-hydroxytryptamme) was the best and tryptophan the worst electron donor for both peroxidase compounds. The enzymes from superfamily II are more competent in substrate oxidation compared with horseradish peroxidase (HRP). HRP compound II is about 10 times less reactive than compound I. On the contrary (with the exception of serotonin), compound II of human peroxidases is shown to be 103-104 times less reactive than compound I. This may be indicative that important structural changes can occur at the substrate binding site when LPO or MPO compound I is converted to compound II. In contrast to peroxidases from superfamily I, in human and animal enzymes (which are thought to be involved in halide and thiocyanate oxidation in vivo) the heme is covalently linked to the protein and the active site residues are different in both orientation and H-bonding.


Characterization of AFT2, a putative iron binding transcription factor in the yeast Saccharomyces cerevisiae.

Shulamit Jaron ~, Julian C. Rutherford a, Dennis R. Winge ~. " Department of Hematology, University of Utah, 50 N Medical Drive, 84132, Salt Lake City, UT,

USA

Iron homeostasis in the yeast Saccharomyces cerevisiae appears to be partially controlled by the gene product of AFT1. Aftlp is a transcription factor, which activates genes responsible for iron uptake in response to low iron levels. An AFT1-1 up mutation results in the activation of the AFT1 regulon, regardless of iron concentrations. A single mutation of the Cys291 to Phe confers the up phenotype. Because Cys291 is within a CXC motif, an appealing hypothesis is that this motif in AFT1 is involved in iron sensing and may, in fact, bind iron.

A search of the yeast database reveals a second ORF encoding a protein with 39% identity to the N-terrmnus of AFT1, including the CXC motif. We have designated this gene AFT2. The regulon of AFT2 has been identified by microarray analysis, and appears to include iron uptake proteins also activated by AFT1. Recent data indicate that iron levels influence transcriptional regulation by AFT2, in a similar manner to AFT1. These data have led to the proposal that AFT2 may also bind iron, and therefor sense cellular iron by a similar mechanism to AFT1. We have successfully cloned AFT2 and established that the N-terminal region contains the DNA binding domain. Here we report the characterization of the Aft2 protein with an emphasis on the putative iron-binding motif.

D N A and R N A damage by Cu(II)-amikacin complex - a possible element of ototoxicity of aminoglycosides

Matgorzata Jezowska-Bojczuk a Wojciech Szczepanik ", Wojciech Bal a, Wojciech Le~niak a, Jerzy Ciesiotka b Jan Wrzesifiski b "Faculty of Chemistry, University of Wroctaw, F. Joliot-Curie 14, 50-383 Wroctaw, Poland blnstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna/t, Poland

Amikacin is a semisynthetic aminoglycosidic antibiotic, a derivative of kanamycin A, having the B 1 amino group of 2-deoxystreptamine moiety modified by acylation with 4-amino-2-hydroxybutyric acid. Our previous study demonstrated that amikacin is a strong chelator for Cu(II) ions.I

Copper(II) could be available to xenobiotic ligands locally under specific conditions, like inflammation. This physiological state involves abundant generation of hydrogen peroxide and oxygen radical species by macrophages. The ability of Cu(II) complexes of aminoglycosides to cleave DNA and RNA was shown recently. 2 Here we present the interactions of Cu(II)-Ami with oxidation-susceptible biomolecules: 2'-deoxyguanosine (dG) leading to its promutagenic 8-oxo derivative, pBR322 plasmid DNA and yeast tRNA Ph~ in both presence and absence of hydrogen peroxide, as well as the mechanism of this activation, involving generation of hydroxyl radicals. 3 Some of these reactions may play a role not only in aminoglycosides bactericidal activity, but also in their oto- and nephrotoxicity.

H2N OH

"° - - . ,Z cu / ",, / o-'%~ , ~ \

HO t OH

1. Jezowska-Bojczuk M., and Bal W. (1998) J. Chem. Soc. Dalton Trans. 153-159. 2. Sreedhara A. and Cowan J. A. (1998) Chem. Commun. 1737-1738, Sreedhara A.,

Patwardhan A. and Cowan J. A, (1999) Chem. Commun. 1147-1148. 3. Jezowska-Bojczuk M., Legniak W., Bal W., Koz|owski H., Gatner K., Jezierski A., Sobczak J., Mangani S., and

Meyer-Klaucke W. (2001) Chem.Res.Toxicol., in press.

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