abstracts poster presentations (z3)

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Journal of lnorganic Biochemistry 86 (2001) 391 Small non-heme iron proteins in a novel superoxide defense system Patficia Raleiras a, Carla Ascenso a, Frank Rusnak b, Isabel Moura a and Jos6 J. G. Moura" "Centro de Quimica Fina e Biotecnologia, Departamento de Quimica, Faculdade de Cirncias e Tecnologia, Universidade Nova de Lisboa, 2829-5 l 6 Monte de Caparica, Portugal (e-mail. patricia, [email protected], unl.pt) ~Section Hematology Research, Mayo Clinic, Rochester, MN 55905, USA Neelaredoxin (Nlr) ~s a small non-hemic iron protein found in several strict anaerobic or microaerophilic microorganisms u. Its iron center contains one atom coordinated by four histidines and one cysteine (see figure), a type of center previously found only in desulfoferrodoxin (Dfx)3; a sixth ligand, a glutamate, was also \ found coordinating the iron atom 4. The glutamate residue and, more recently, a nearby lysine "~&(',\~ ~) [k~\ residue, have been proposed to have a specific role in this protein's activity 5. Nlr has been " ~'"" \" ..... characterized as a superoxide reductase, a novel type of oxidative stress defense system, by "~k,] which O-2 doesn't suffer dismutation (1) but only reduction (2): (1) 2 0"2 + 2H + ~ H202 + N/" \ "X~ O, (2) O-z + 2H + ~ H202 _.~'~/~' b, Dfx also contains another iron center resembling desulforedoxin (Dx), a small protein found ~- "'~~.~ \k~\~.,, only in Desulfovibrio gigas, its role remaining unclear. Also Nlr's electron donor hasn't been found. We are currently working with various techniques, including enzymatic assays, computerized protein docking simulation and electrochemistry, to fmd this electronic partner, which could be Dx rubredoxin (as proposed for Nlr from Pyrococcus furiosus) or other small electron transfer protein. This work discusses the Dfx's Dx-like center role. 1. T.Jovanovi'c et al. J. Biol.Chem. 275, 28439 (2000) 2. L. Chen, P. Sharma, J. Le Gall, A. M. Mariano, M. Teixeira, and AN.Xavier, Eur.J.Biochem. 226, 613 (1994) 3. I.Moura, P.Tavares, J.J.G.Moura, N.Ravi, B.H.Huynh, M.-Y.Liu, J.LeGall, J.Biol.Chem. 265, 21596 (1990) a. A. P. Yeh, Y. Hu, F. E. Jenney, Jr., M. W. W. Adams, and D. C. Rees, Biochemistry 39, 2499 (2000) 5. M. Lombard, C. Houre-Levin, D. Touati, M. Fontecave, V. Nivibre, Biochemistry 40, 5032 (2001) PRAATS-FCT-MCT supports this work. Study of the interaction of vanadyl ion with the ligand 1,2-dimethyl-3-hydroxy-4- pyridinone Mafia Rangel a, Ricardo Pintob, Paula Gameiro b, Eul/dia Pereira b, Baltazar Castro b and John Burgess c " CEQUP, Instituto de Cirncias Biomrdicas Abel Salazar 4099-003 PORTO, Portugal(email: m crangel@fc, up.p 0 CEQUP, Departamento de Quimica, Faculdade de Cidncias do Porto, 4169-007 PORTO, Portugal Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK The insulin-like action of pyridinone oxovanadium(IV) complexes has been revealed on in vitro studies concerning the inhibition of FFA release in adipocytes L and the knowledge of the chemical properties of these systems in different chemical environments is crucial for the understanding of their action and the development of oral substitutes of insulin. In this work we report the results obtained in aqueous solution under anaerobic conditions for the compound bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)- oxovanadium(IV) and solutions of vanadyl sulphate and the free ligand prepared in variable metal:ligand ratios and at variable pH. ~ ~ The work has been performed using EPR spectroscopy and potentiometric and /.-.. ~o ~ ..s-..c., voltammetric methods. The formation constants of complexes and speciation diagrams ,,,c-~ ...... "-¢ '-o" v\d';'!" "-''j are presented as well as the spin-hamiltonian parameters of the species in solution. .,,: 1. Rangel M., Tamura A., Fukushima C., Sakurai H., J. Biol. Inorg. Chem., 6, 128 - 132 (2001)

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Page 1: Abstracts Poster Presentations (Z3)

Journal of lnorganic Biochemistry 86 (2001) 391

Small non-heme iron proteins in a novel superoxide defense system

Pat f ic ia Ra le i ras a, Ca r l a A s c e n s o a, F r a n k R u s n a k b, Isabel M o u r a a and Jos6 J. G. Moura"

"Centro de Quimica Fina e Biotecnologia, Departamento de Quimica, Faculdade de Cirncias e Tecnologia, Universidade Nova de Lisboa, 2829-5 l 6 Monte de Caparica, Portugal (e-mail. patricia, [email protected], unl.pt) ~Section Hemato logy Research, Mayo Clinic, Rochester , MN 55905, USA

Neelaredoxin (Nlr) ~s a small non-hemic iron protein found in several strict anaerobic or microaerophilic microorganisms u. Its iron center contains one atom coordinated by four histidines and one cysteine (see figure), a type of center previously found only in desulfoferrodoxin (Dfx)3; a sixth ligand, a glutamate, was also

\

found coordinating the iron atom 4. The glutamate residue and, more recently, a nearby lysine "~&(',\~ ~) [k~\ residue, have been proposed to have a specific role in this protein's activity 5. Nlr has been " ~'"" \" ..... characterized as a superoxide reductase, a novel type of oxidative stress defense system, by "~k,] which O-2 doesn't suffer dismutation (1) but only reduction (2): (1) 2 0"2 + 2H + ~ H202 + N/" \ "X~ O, (2) O-z + 2H + ~ H202 _.~'~/~' b, Dfx also contains another iron center resembling desulforedoxin (Dx), a small protein found ~- "'~~.~ \k~\~.,, only in Desulfovibrio gigas, its role remaining unclear. Also Nlr's electron donor hasn't been found. We are currently working with various techniques, including enzymatic assays, computerized protein docking simulation and electrochemistry, to fmd this electronic partner, which could be Dx rubredoxin (as proposed for Nlr from Pyrococcus furiosus) or other small electron transfer protein. This work discusses the Dfx's Dx-like center role.

1. T.Jovanovi'c et al. J. Biol.Chem. 275, 28439 (2000) 2. L. Chen, P. Sharma, J. Le Gall, A. M. Mariano, M. Teixeira, and AN.Xavier, Eur.J.Biochem. 226, 613 (1994) 3. I.Moura, P.Tavares, J.J.G.Moura, N.Ravi, B.H.Huynh, M.-Y.Liu, J.LeGall, J.Biol.Chem. 265, 21596 (1990) a. A. P. Yeh, Y. Hu, F. E. Jenney, Jr., M. W. W. Adams, and D. C. Rees, Biochemistry 39, 2499 (2000) 5. M. Lombard, C. Houre-Levin, D. Touati, M. Fontecave, V. Nivibre, Biochemistry 40, 5032 (2001) PRAATS-FCT-MCT supports this work.

Study of the interaction of vanadyl ion with the l igand 1,2-dimethyl-3-hydroxy-4- pyridinone

Mafia Rangel a, Ricardo Pinto b, Paula Gameiro b, Eul/dia Pereira b, Baltazar Castro b and John Burgess c " CEQUP, Ins t i tu to de Cirncias B iomrdicas Abel Salazar 4099-003 PORTO, Por tuga l (emai l :

m crangel@fc, up.p 0 CEQUP, Depar tamen to de Quimica, Facu ldade de Cidncias do Porto, 4169-007 PORTO, Por tugal Depar tmen t o f Chemis try , Univers i ty o f Leicester , Le ices ter LE1 7RH, UK

The insulin-like action of pyridinone oxovanadium(IV) complexes has been revealed on in vitro studies concerning the inhibition of FFA release in adipocytes L and the knowledge of the chemical properties of these systems in different chemical environments is crucial for the understanding of their action and the development of oral substitutes of insulin.

In this work we report the results obtained in aqueous solution under anaerobic conditions for the compound bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)- oxovanadium(IV) and solutions of vanadyl sulphate and the free ligand prepared in variable metal:ligand ratios and at variable pH. ~ ~

The work has been performed using EPR spectroscopy and potentiometric and /.-.. ~ o ~ ..s-..c., voltammetric methods. The formation constants of complexes and speciation diagrams ,,,c-~ . . . . . . "-¢ '-o" v \d ' ; ' ! " "-''j are presented as well as the spin-hamiltonian parameters of the species in solution. .,,:

1. Rangel M., Tamura A., Fukushima C., Sakurai H., J. Biol. Inorg. Chem., 6, 128 - 132 (2001)

Page 2: Abstracts Poster Presentations (Z3)

392 Journal of lnorganic Biochemistry 86 (2001)

Malleability of a catalytic zinc site: inverting the role of Zn2 in b.cereus metallo-~- lactamase

Rodolfo M. Rasia, Alejandro J. Vila. Biophysics section, Department of Biological Chemistry, University of Rosario. Suipacha 531, S2002LRK, Rosario, Argentina. e-mail. rrasia@fbioyf unr. edu.ar.

[3-1actamases are hydrolases which cleave the amide bond of [3-1actam antibiotics, confering resistance to microorganisms which express them. Class B ]3-1actamases are metalloenzymes, which require Zn(II) for their activity. The metallo-13-1actamase family includes both binuclear and mononuclear enzymes. Bacillus cereus metallo-D-lactamase ([3LII) is unique among these because the mono-zinc(II) (Znl) form is functional, and the binuclear enzyme (ZniZn2) has an enhanced hydrolytic capability. Zn2 is thus a nonessential activator in [3LII.

We have engineered potential His and Glu ligands by mutating residue Arg91, located in the active site. A His is present in this position in the homologous metallo-[3-1actamase L 1 from S.maltophilia, being a ligand of Zn2. Both mutant enzymes are active in their mononuclear form but, in contrast to wild type [3LII, Zn2 acts as a mixed inhibitor. The UV- visible spectra ofbi-cobalt(II) (CojC02) and mixed zinc(II)-cobalt(lI) (ZniC02) substituted [3LII mutants reveal changes in the coordination environment of the second binding site. The Zn-edge XANES spectra indicate that the Zn~ site is not disturbed by the mutation. Paramagnetic NMR spectra of the cobalt(II) adducts suggest the presence in the second metal site of a single His ligand in R91H [3LII and no His ligand in R91E ]3LII. In toto, spectroscopic data reveal that the mutations have altered the Zn2 coordination environment (i.e. the non-essential zinc site). We propose that the inhibition is due to a conformational rearrangement of Asp90 (ligand of the Zn2 site), that seems to be essential for the enzyme activity.

UNR, CONICET, ANPCyT and Fundaci6n Antorchas are acknowledged for financial support. RMR thanks CONICET for a graduate fellowship.

Ca 2+ binding to high affinity sites of proteins measured by capillary electrophoresis

Bodil W. Rasmussen, Morten J. Bjerrum Bioinorganic Group, Department of Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark (email." [email protected])

The stability constant for the binding of metal ions to a protein is most often determined using classical methods such as spectrophotometry, ultrafiltration or equilibrium dialysis. Those methods all require a high amount of the compounds to be investigated, which is often a problem in the investigation of proteins. Analysis of Ca 2÷ binding to Ca 2÷- containing proteins represents an additional problem, since Ca 2÷ is present in even high purity water, making the elucidation of binding constants > 10 5 M ~ a very uncertain and difficult task.

We present a new method for analysis of protein-metal binding, based on capillary electrophoresis, that utilizes the change in electrophoretic mobility of the protein upon binding of a metal ion. The electrophoretic mobility is measured in solutions that are as identical as possible with the exception of the concentration of the free metal ion in question.

Taking advantage of EDTA (1,2-diaminoethane-N,N,N',N'-tetraethanoic acid) and DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraethanoic acid) as controllers of the Ca 2+ concentration, a [Ca2+]nee as low as l0 -12 M has been attained in the buffer solutions. We have found this method a preferable tool for measuring binding of Ca 2. or other metal ions to proteins, where the binding constant is in the range 10 4-10 ~ M ~. Another advantage of this method is the very small sample volume that is needed.

The stability constants for the binding of Ca 2+ to Horseradish Peroxidase and Bovine c~-lactalbumin has by this new method been measured to 10104 M ~ and 10 6.8 M -I respectively at an ionic strength of 0.1, pH 7.4. and 25°C.

Page 3: Abstracts Poster Presentations (Z3)

Journal of Inorganic Biochemistry 86 (2001) 393

Understanding the catalytic centre, Cuz, of nitrous oxide reductase by spectroscopy

Tim R a s m u s s e n a,b, Ben C. Berks a, and Andrew J. Thomson b Centre for Metal loprote in Spec troscopy and Biology, Schools o f Biological ~ and Chemical b Science, Universi ty o f E a s t Anglia, Norwich ,NR4 7T J, UNITED KINGDOM (e-mail." t. rasmussen@uea .ac .uk )

Some bacteria use oxidised forms of nitrogen as electron acceptors to survive under anaerobic conditions by denitrification. The last step of denitrification, the two-electron reduction of nitrous oxide to dinitrogen, is catalysed by the multi-copper enzyme nitrous oxide reductase. Each subunit of a functional homodimer has a binuclear copper centre, CUA, as an electron transfer group, and a novel tetranuclear copper centre, Cuz, as the catalytic site ~. Different spectroscopic methods (UV/Vis, MCD, EPR, RR) and inorganic sulfur determinations showed that Cuz has a central bridging sulfur atom as ligand 2 which was confirmed by the X-ray crystal structure 3. Cuz is therefore the fncst example of a copper-sulfide centre in biology. The spectra are analysed to understand the electronic structure of Cuz. Of five theoretically possible redox-states of Cuz only two, interconverted by one electron, have been detected by spectroscopy. In another enzyme form, isolated when the enzyme is incubated with oxygen, Cuz is fixed in one redox-state although the enzyme shows normal steady-state activity. These results are interpreted in terms of a catalytic mechanism. [Support is from the UK BBSRC]

1. Brown, K., Tegoni, M., Prudencio, M., Pereira, A.S., Besson, S., Moura, J.J., Moura, I., and Cambillau, C. Nature Struct. Biol. 7, 191-195 (2000).

2. Rasmussen, T., Berks, B.C., Sanders-Loehr, J., Dooley, D.M., Zumft, W., and Thomson, A.J. Biochemistry 39, 12753-12756 (2000).

3. Brown, K., Djinovic-Carugo, K., Haltia, T., Cabrito, I., Saraste, M., Moura, J.J., Moura, I., Tegoni, M., and Cambillau, C. J. Biol. Chem. 275, 41133-41136 (2000).

Thiolate oxygenation in N-carboxamido and S-thiolato Co(Ill) complexes related to nitrile hydratase metallic active site.

Mathieu Rat, Rodolphe Alves de Sousa, Sandrine Chatel, Daniel Mansuy and Isabelle Artaud Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques. UMR 8601-

CNRS, UniversitO RenO Descartes, 45 rue des Saints-POres 75270 Paris cedex 06, France (e mail. mathieu rat@yahoo. fr )

Nitrile Hydratases (NHases) are non-heme iron or non-corrinoid cobalt enzymes wich catalyse the hydration of nitriles to amides. Recent X-Ray studies on a Fe-NHase have revealed an unprecedented coordination mode of the iron center : the metal is ligated to two carboxamido nitrogens and three sulfur donors in different oxidation states (namely thiolate, sulfenate R S O and sulfmate RSO2 ). Recent ENDOR studies indicate ,.~ that a hydroxide group or a water molecule occupies the sixth site, hence conferring a low spin six-coordinate iron(III) with pseudooctahedral geometry. EXAFS studies and sequence ~ ~ ~,~ _ homologies suggest also that iron(III) and cobalt(III) NHases should have very similar active a4 sites. As part of our effort toward understanding the selective oxygenation mechanism of the equatorial thiolates, we have synthesized several square planar CON2S2 complexes containing two carboxamido nitrogens and two aliphatic thiols. Very recently, we have reported their oxygenation using dimethyldioxirane in the presence of tertbutylisonitrile t affording six- ~ coordinate sulfinato complexes. We present in this account the reactivity of the CoNzS2 [CoN2(SO2)2(tBuNC)2]- complexes toward axial ligands and their oxidation with different oxygen donors. The structure and spectroscopic properties of disulfinato and sulfmato/sulfenato-Co(III) complexes will be compared and a mechanism for the post-translational oxidation of the equatorial cysteines will be proposed.

1 Rat M., Alves de Sousa R., Vaissermann J., Leduc P., Mansuy D., Artaud I., J. Inorg. Biochem., 84, 207-213 (2001)

Page 4: Abstracts Poster Presentations (Z3)

394 Journal of Inorganic Biochemistry 86 (2001)

C y t o t o x i c i t y a n d X - r a y s t r u c t u r e ana lys i s o f nove l c i sp la t in a n a l o g u e s

Michael J. Rauterkus a, Ina Puscasu a, Christ ian M o c k a, Gesche Tal len b, J. E. A. Wolff b, Bernt Krebs a Anorganisch-Chemisches institut, Westfiilische Wilhelms-Universitiit Miinster, Wilhelm-Klemm- Strafle 8, 48149 Miinster, GERMANY (e-mail: rauterk@nwz, uni-muenster.de) Pediatric Oncology Program, Alberta Children's Hospital, The University of Calgary, 1820 Richmond Rd. SW, Calgary AB, T2T 5C7, CANADA

The antitumor activity of cisplatin arises from its binding to genomic DNA. The majority of cisplatin-DNA adducts (> 90 %) are intrastrand 1,2-d(GpG) and 1,2-d(ApG) crosslinks in which the two chlorine atoms of the cisplatin molecules are replaced by the N7 atoms of adjacent purme bases. These cisplatin-DNA complexes bend and unwind the duplex at the site of damage with its major groove being compressed and the minor groove widened. The clinical success of cisplatin is compromised by various mechanisms of resistance such as repair of cisplatin-modified DNA by enzymatic ~emoval of the adducts. To get more information about structure-activity relationships we have designed several new hydrophobic platinum complexes with cis-geometry of the chlorine atoms which were examined by X-ray structure analysis, as well as lhelr palladium analogues. In a series of investigations we have studied the cytotoxicity of the complexes [Pt(dpea)Cl2], [Pt(dpha)Clz], [Pt(dpm)Cl2], [Pt(dbpa)C12] and [Pt(phen)Cl2], [Pt(nitro)C12], [Pt(oxim)Clz], [Pt(sulfid)Cl2] ~ in the U251 human malignant glioma cell line in vitro. The number of surviving cells was determined by MTT tests. Additionally, the solubility of the complexes in water was examined. As a result a higher cytotoxicity of the more hydrophobic compounds was found. Because of the enormous structural differences of the complexes tested the concept of an unspecific transport mechanism of cisplatin and its analogues though the cell membrane is supported.

1. M611er N., Kangarloo B. S., Puscasu I., Mock C., Krebs B. and Wolff J. E. A., Anticancer Res., 20, 4435 (2000).

The support of the DEGUSSA AG, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.

A c t i v e site a r c h i t e c t u r e in m a m m a l i a n h a e m p e r o x i d a s e s

Emma Lloyd Raven, a Neesha Patel, a Mairtin O'Coinceanainn, a Martin Mewies, a Harold, M. Golf, b Michael L. Ott. b " Department of Chemistry. University of Leicester, University Road, Leicester, LE1 7RH,

England, UK. b Department of Chemistry, University of Iowa, lowa City, Iowa 52242, USA.

Mammalian peroxidases, examples of which includes myeloperoxidase (MPO) and lactoperoxidase (LPO), catalyse reactions of crucial importance to a wide range of biological processes. The active site structure of this class of haem is distinct from the classical haem b-containing peroxidase enzymes, and involves covalent ester linkages between modified methyl groups on the haem and carboxylate groups on the protein, Figure 11-3; the MPO enzyme also contains a unique sulphonium linkage, which has not been observed in any other peroxidase enzyme. Using site-directed mutagenesis, we have engineered new covalent linkages, analogous to those found in MPO and LPO, into the active site of recombinant pea cytosolic ascorbate peroxidase (APX) by placement of methionine (S 160M variant) and aspartic acid ($207D variant) residues close to the haem 2-vinyl and 1-methyl groups, respectively. Characterisation of these variants, and the effect on the spectroscopic properties and biochemical reactivity of APX have been assessed.

H~C /Mel S" f

CH~CH

GluCOCH 2 1 ~

H2(~Asp HOOC COOH

Figure 1. The active site of MPO.

1. Fielder, T.J. ,Davey, C.A.,Fenna, R.E. ,J . Biol. Chem.,275, 11964-11971 (2000) 2. Kooter, I. M., Pierik, A. J., Merkx, M., Averill, B. A., Moguilevsky, N., Bollen, A., Wever, R. J. Am. Chem. Soc..

119, 11542-11543 (1997) 3. Rae, T., Goff, H. M. J. Am. Chem. Soc. 118, 2103-2104 (1996) Financial support from The Wellcome Trust and EPSRC are gratefully acknowledged.

Page 5: Abstracts Poster Presentations (Z3)

Journal of Inorganic Biochemistry 86 (2001) 395

A density functional study of cisplatin interaction with nucleobases

Nazzareno Re and Alessandro Marrone Facolta' di Farmacia, Universita' degli Studi G. D'Annunzio, Via Dei Vestini, ]3, 1-06100 Chieti, italy

Since discovery of the antitumor activity of cis-dichloro-diammineplatunum(II) (cis-DDP) a considerable amount of research has focused on elucidating the mechanism of activity of this drug.l A large body of evidence has soon indicated that cls-DDP binds to the nitrogen atoms of the DNA nucleobases. Following these studies, there has been considerable interest in the interaction of cis-DDP with DNA and its purine and pyrimidine constituents.: In this work, density flmctional calculations have been performed on cis-[Pt(NH3)2C1B] + complexes, where B is an adenine, guanine, citosine, thymine or uracile nucleobase. Calculations were based on a Becke and Perdew exchange-correlation potential using triple-~ plus polarization basis sets and include scalar relativistic corrections. Geometry optimizations have been performed for all possible coordination modes between the cis-[Pt(NH3)2C1] ~ fragment and the nucleobases. Bond energies have been calculated for all the considered metal binding sites. Our results allow to built a binding order for the nitrogen atoms of the considered nucleobases and indicate that the N7 atom of guanine is the thernaodinamically preferred coordination site. I~ Lippert B., Coord. Chem. Rev., 200-202, 487-516 (2000) 2 Jamieson E. R. and Lippard S. J..Chem. Rev., 99, 2467-2498 (1999)

Molecular and kinetic characterization of cyanobacterial iron- and manganese- containing superoxide dismutases

Guenther Regelsberger a, Ulrike Laaha a, Dagmar Dietmann a, Florian Rueker b, Guenter A. Peschek c, and Christian Obinger a "Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-l~90 Vienna,

AUSTRIA (e-mail. [email protected]) ~!nstitute Of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A- 1190 Vienna, AUSTRIA ~lnstitute of Physical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna,

AUSTRIA

Superoxide radical anions are produced as byproducts of normal metabolism and, especially in cyanobacteria, in oxygenic photosynthesis, To prevent cell damage by destruction of iron-sulfur proteins and formation of highly reactive hydroxyl radicals, superoxide dismutases are necessary to lower the steady state concentration of superoxide within cells. We used the nitrogen-fixing cyanobacterium Anabaena variabilis to study their superoxide disnmtases (SODs). Kinetic measurements showed that both membrane preparations and cytosolic extracts exhibit SOD activity. Western blotting and immunodetection applying antibodies against iron- and manganese-SOD depicts that a FeSOD is in the cytosol and a MnSOD is membrane-bound. Their localization was also confh-med within vegetative cells and heterocysts by immunogold labeling and transmission electron microscopy. Both enzymes were cloned, sequenced and expressed in E. coli. The FeSOD is an acidic, dimeric enzyme (23 kDa per subunit) and the specific activity was approximately 2000 units per mg. Whereas it is not inhibited by cyanide, it is inhibited by azide and irreversibly inactivated by hydrogen peroxide. Overexpression of the whole Mn-SOD was not successful; this may be due to the hydrophobic domain located at the N- terminus that is proposed to act as a membrane anchor. So a soluble portion of the Mn-SOD without the membrane anchor was expressed and purified. Stopped-flow spectroscopy was applied to follow the reaction of both SODs with superoxide at 245 nm to determine kinetic parameters.

Page 6: Abstracts Poster Presentations (Z3)

396 Journal of Inorganic Biochemistry 86 (2001)

Redox and acid base propert ies of the n ine -haem cy tochrome c from desul fovibrio desul fur icans A T C C 2 7 7 7 4

Clara C. Reis, Ricardo O. Louro, Ant6nio V. Xavier

Instituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, 2780-156 Oeiras, PORTUGAL (e-mail: [email protected])

The nine-haem cytochrome (9Hcc), isolated from the sulfate reducing bacteria (SRB) Desulfovibrio desulfuricans ATCC 27774 (Dd ATCC 27774), is a 37.8 kDa monomeric protein composed of two tetrahaem cytochrome c3 domains with one extra haem located between them ~. Its role in the bioenergetics metabolism is possibly analogous to that of the high molecular weight cytochrome (Hmc) containing sixteen haems found in the SRB D.vulgaris as it shows a high homology to the C terminal of Hmc and such a protein was never found in Dd ~.The electrons and protons generated upon oxidation of molecular hydrogen by hydrogenase are received by the periplasmic tetrahaem cytochrome c3. The 9Hcc can accept electrons from cytochrome c3 via specific docking 2 and acts as electron carrier across the membrane. Kinetic reduction experiments showed a 5-fold increase in the reduction rate of 9Hcc in the presence of c3 t. In order to study further the functional properties of 9Hcc, redox titrations followed by visible spectroscopy were performed at various pH values in the range of 6.3 to 8.4 showing a pH dependence of the reduction potentials of the haems (redox-Bohr effect). This data provides further support to the proposal that 9Hcc participates in the energy transduction. In order to obtain a more detailed characterisation of these phenomena, analysis of the NMR spectra of the oxidised form is currently under way.

1. Matias, P.M., Coelho, R., Pereira, I.A.C., Coelho, A.V.,Thompson, A.W., Sieker; L.C., LeGall, J., Carrondo, M.A, Structure 7:119-130 (1999). 2. Matias, P.M., Saraiva, L.M., Soares, C.M., Coelho, A.V., LeGall, J., Carrondo, M.A.J. Biol. Inorg. Chem. 4:478-

494. (1999).

This work was funded by EU contract ERBFMRX-CT98-0218.

Carboxy la t ion of a lkanes catalyzed by v a n a d i u m c o m p o u n d s

Patricia M. Reis a, Jos6 A.L. Silva a, Ant6nio M.F.Palavra a, Jo~o J.R. Frafisto da Silva a, Yuzo Fujiwara b and Armando J.L. Pombeiro a

Centro de Quimica Estrutural, Complexo l, Instituto Superior TOcnico, Av. Rovisco Pais, 1049- 001, Lisboa, Portugal (e-mail: patricia.reis@popsrv, ist. utl.pt)

b Henkel Reserch Center of Advanced Technology, Molecular Engineering Institute, Kinki University, 11-6 Kayanomory Iizuka, 820-8555, Fukuoka, Japan

Activation of alkanes by transition metal centres and their conversion into more valuable products such as acids, alcohols, ketones,etc, have attracted great attention recently. In pursuit of our interest on the activation of small molecules by vanadium complexes 1'2, we are now reporting that a series of vanadium(IV) or (V) complexes of polydentate ligands such as triethanolaminate or N-hydroxyiminodiacetate act as efficient catalysts, in the presence of peroxodisulfate and in acidic medium, for the carbonylation (by carbon monoxide) of saturated hydrocarbons [both linear (e.g. pentane or hexane) and cyclic ones (e.g. cyclohexane)], towards the corresponding carboxylic acids. The selectivity and activity of the V-catalysts under various experimental conditions will also be presented.

1. Silva M.F.C.G., Silva J.A.L., Silva J.J.R.F., Pombeiro A.J.L., Amatore C., Verpeaux J.-N., J.Am.Chem.Soc., 118, 7568-7573 (1996) 2. Reis P.M., Silva M.F.C.G., Silva J.A.L., Silva J.J.R.F., Pombeiro A.J.L., Chem. Commun., 1845-1846 (2000)

This work has been partially supported by the Foundation for Science and Technology (FCT) and the PRAXIS XXI Programme (Portugal).

Page 7: Abstracts Poster Presentations (Z3)

Journal of Inorganic Biochemistry 86 (2001) 397

Hydrogen peroxide-specific inactivation of calcineurin in T-lymphocytes

T i f f a n y A. Re i t e r a, F r a n k R u s n a k a

'~ Sect ion o f Hemato logy Research and Depar tmen t o f B iochemis t ry and Molecu lar Biology, Mayo Clinic and Foundat ion , 200 Firs t S t ree t SW, 55905, Rochester , U.S.A. (e-mai l . reiter, t i f fany@mayo, edu)

Calcineurin is a member of the serine/threonine phosphatase family and contains an active site dinuclear metal cluster ( ~ 1 equivalent each of Fe and Zn). Activated calcineurin in T-lymphocytes dephosphorylates a transcription factor termed N-F-AT. Dephosphorylated NF-AT undergoes nuclear translocation and results in transcriptional activation of the IL-2 (interleukin-2) gene. The active site of calcineurin is nearly identical to the active site of purple acid phosphatase; a diiron, redox sensitive enzyme. Mammalian PAP and calcineurin metalloisoforms, Fe-Fe and Fe-Zn, are active only in the mixed valent oxidation state in vitro. To monitor intracellular calcineurin activity we use two NF- AT/luciferase reporter plasmids.

We have shown that calcineurin is sensitive to H202 in vivo (ICs0 _. 30 gM), yet insensitive to superoxide (1 mM paraquat) and hypochlorite (600 pM). Nevertheless, calcineurin is sensitive to superoxide (ICs0 <60 ~tM), hypochlorite (ICs0 150 gM), and H202 (IC50 5 70 gM) in vitro. Cells treated with H202 and NaOC1 for up to 1 hour do not undergo a change in cellular redox potential as measured by assessing [GSH]/[GSSG] ratios. The reduction in calcineurin activity is evident in cells up to 6 hours after initial H202 exposure, despite the short lifetime of H202 in culture media (< 5 min). Calcineurin inactivation in brain cell lysate follows very rapid kinetics (inactivation within 10 seconds of H202 addition). From these studies we conclude that the mechanism of calcineurin inactivation in T-cells is specific for H202 and appears to involve a direct redox reaction with the enzyme.

1. Reiter T.A., Abraham R.T., Choi M., and Rusnak F., J. Biol. Inorg. Chem., 4,632-644 (1999) 2. Rusnak F. and Reiter T., Trends Biomed. Sci., 25,527-529 (2000)

Supported by NIH grant GM46865 (to F.R.)

A new general molecular mechanics force field for the oxidized form fo blue coppper proteins

Rainer Remengi, Peter Comba A n o r g a n i s c h - C h e m i s c h e s Insti tut , Univers i td t Heidelberg, INF 270, D-69120 Heide lberg Fax. ÷49-6221-54-661 7,( e -mai l . r emeny i@akcomba .oc i , un i -he ide lberg ,de)

Blue Copper Proteins exhibit extraordinary fast electron transfer, unusual redox potentials and spectroscopic properties.Ill One of the most important tasks in the investigation of this class of proteins is, to find out about the origin of these properties and to correlate them to structural features of the metal site. However, until now a lot of approaches have been done, but most of them failed due to the inaccuracy of experimental structural data.[ 2] The aim of this work was to develop a general force field, valid for all oxidised blue copper proteins on the basis of the AMBER[3] force field. No experimental data was used to develop the parameters for the metal site, but a DFT/B3LYP study of a theoretical model for the metal site was used for this purpose. After a study of several blue copper proteins we can show, that the force fiel produces reliable optimised structures. The structures correlate well with UV/Vis spectroscopy as it is known from theoretical work.[ 4] 1. Comba, P. Coord. Chem. Rev, 200-202, 217-245 (2000). 2. Fields, B. A.; Bartsch, H. H.; Bartunik, H. D.; Cordes, F.; Guss, J. M.; Freeman, H. C. Acta Crystallogr., D50, 709-

730 (1994). 3. Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.; Cheatham, T. E.; Debolt, S.; Ferguson, D.; Seibel, G.;

Kollman, P. Comput. Phys. Commun., 91, 1-41 (1995). 4. Pierloot, K.; DeKerpel, J. O. A.; Ryde, U.; Olsson, M. H. M.; Roos, B. O. J. Am. Chem. Soc., 120, 13156- 13166

(1998).

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398 Journal of Inorganic Biochemistry 86 (2001)

Novel cyclic iron(Ill) pivalates with fluoro and/or alkoxo bridges

E v a Ren t sch l e r b, G r i g o r e A. T i m c o a, T h o m a s W e y h e r m t i l l e r b, , V l a d i m i r B. A r i o n c, Andre i S. B a t s a n o v d

"Academy of Sciences, Institute of Chemistry, Academiei Str., MD-2028 Chi4in~u, R. Moldava bMax Planck Institut fiir Strahlenchemie, Stiftstrasse 34-36, 45470 Miilheim, Germany ~Institut fiir Anorganische Chemic der Universitiit Wien, Wiihringerstr. 42, A-I090 Wien, Austria aDepartment of Chemistry, Durham University, South Road, Durham DH1 3LE, United Kingdom

The synthesis of "ferric wheels" has received a great deal of attention in the last years due to their relevance to biological systems, magnetic imaging chemistry and single-molecule magnets. A variety of high-nuclearity cyclic iron compounds containing oxo-, hydroxo- and/or alkoxo- bridged metal ions have been synthesised. In contrast, the number of well-documented fluoro bridged iron(III) complexes is scarce. We report on the synthesis of a number of fluoro- bridged iron(III) wheel compounds, their conversion into mixed alkoxo-/fluoro- bridged or purely alkoxo- "bridged ring structures. Starting from FeF33H20 and pivalic acid in the presence of R2NH (R - lso-Pr, cyclohexyl, Et, n-Pr, n-Bu) both cavity-vacant FesFsPiVl6 (I) and amine-incorporating octanuclear ferric wheel compounds FesFsPiv~6R2NH, where R - Et (II), n-Pr (III), n-Bu (IV), have been prepared. Compound I, in contrast to II-IV, can accommodate various neutral guest molecules inside the cavity. The fluoro bridges in the vacant-cavity I can be completely or partially replaced by alkoxo-groups on reaction with an alcohol, which is accompanied by ring expansion leading to ferric wheels with 10 iron atoms. Thus, the reaction with methanol yielded Felo(OCH3)20Pivl0 (V) and those with ethanol or n-propanol led to FeloFs(OR)lsPivlo with R = Et (VI) and n-Pr (VII). The reaction of I with PhOH gave rise to FesF4(OPh)sPivl2 (VIII). We discovered that upon hydrolysis a part of the fluoride ions can be replaced by hydroxo ligands with the preservation of the wheel structure or with conversion into a complex with the g3-0xo iron triangle core. The latter can be rearranged into Fe~2(OCH3)_,4Piv~2 (IX) when treated with methanol. All compounds were studied by X-ray diffraction methods, M6ssbauer spectroscopy and magnetic susceptibility measurements. The results obtained will help to develop new strategies for designing more sophisticated molecules with rotaxane and catenane structures.

Trinuclear pivalate bridged metal complexes as versatile building blocks for a wide variety of oligonuclear compounds

Eva R_ en tsch le r a, T h o m a s W e y h e r m t i l l e r a and Gr igo re A. T i m c o b

"~Max Planck institut fiir Strahlenchemie, Stiftstrasse 34-36, 45470 Miilheim, GERMANY (e-mail: [email protected], de bAcademy of Sciences of the Republic of Moldava, Institute of Chemistry, str. Academiei, 3, MD-

2028 Chi~in~u, MOLDA VA

The trinuclear iron (III) complex [Fe3(P.3-O)(piv)6(H20)3]piv was shown to be a versatile starting material in the preparation of a variety of oligonuclear iron complexes. In strong dependence of the reaction conditions atleast three different hexanuclear iron (III) compounds can be isolated. Without the use of solvents capable to coordinate to transition metal ions the hexanuclear compound [Fe6(g3-O)2(g2-OH)z(pivh2] 1 is formed. Two Fe30 units are connected via two bridging hydroxo groups which are located above and below the coordination mean plane of the iron atoms. Whereas the inner iron atoms are six coordinated, the two outer iron ions are in an uncommon five-coordinated environment as depicted in the scheme below. In the presence of additional coordinating ligands, the geometrical parameters of the Fe30 core change. The bridging pivalato anions rearrange and in consequence the hydroxo groups can accommodate on the same side of the iron plane, thus resulting in a cis configuration for complex [Fe6(g3-Oh(gz-OH)2(piv)12L2] 2. Whereas compound 1 does not react with H202, compound 2 is easily oxidized to a bexanuclear iron-peroxo species. [Fe6(g3-O)z(p.2-O2)(pivh2L2 ] 3 can be obtained in high yields. Therefore we have been successful to perform accurate magnetic measurements as well as applying M6ssbauer spectroscopy. For all obtained compounds, the differences in the electronic properties, investigated by magnetic susceptibility measurements and been correlated to the structural changes.

2

M6ssbauer spectroscopy, have

1. Banci L., Bertini I., Ferroni, F. and Rosato, A., Eur. J. Biochem., 249, 270-279 (1997) The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.

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Journal of lnorganic Biochemistry 86 (2001) 399

Aryl-Cu(III ) complexes: an intermediate in aromatic hydroxylat ion

Xavi Ribas a, Bruno Donnadieu b, Deanne Jackson ~, Keith O. Hodgson ~, Britt Hedman d, T. Daniel.P. Stack ~ , Antoni Llobet a ~' Dep. Quimica, Facultat de Cikncies, Universitat de Girona, E-17071 Girona. SPAIN (e-mail. [email protected]) ~'Laboratoire de Chimie de Coordination, UPR CNRS 8241, F-31077 Toulouse, FRANCE

Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA ,t Stanford Synchrotron Radiation Laboratory, SLAC, Stanford University, Stanford CA 94309, USA

Metal-mediated activation of C-H bonds is an important step in many industrial and biological oxidation reactions, and elucidation of the mechanism of these processes will inevitably allow better catalyst design. A series of Cu(II) complexes with closely related triazamacrocyclic ligands will be presented that exhibit activation of an aromatic C-H group within each ligand through a quantitative Cu(II) disproportionation reaction at 298 K to yield unprecedented aryl-Cu(III) complexes. These Cu(III)-aryl species are thermally stable in protic media and lack distinctive optical LMCT bands. Crystallographic characterization and XAS spectroscopy confirms the trivalent oxidation state for several different complexes. Each of these Cu(III)-aryl species undergoes aromatic hydroxylation upon addition of aqueous base to form a well-characterized phenoxo-Cu(II) complex (~65%). This hydroxylation proceeds through spectroscopically distinct intermediates that are also present in the reaction of dioxygen with the Cu(I) complexes generated from these ligands. The convergence of these two processes ~hrough similar intermediates and products suggests that the Cu(I)/dioxygen hydroxylation reactions may also proceed through a Cu(III)-aryl species. This Cu(1)/dioxygen aromatic hydroxylation chemistry is reminiscent of tyrosinase, an enzyme that involves a (g:.qZ:.q2_ peroxo)dicopper(II) species in the oxidation process. Through chemical precedent in model chemistry, the involvement of Cu(III) in the oxidation mechanism of tyrosinase has been considered, but not in the form on an aryl-Cu(III) species) Full characterization of these complexes will be presented along with reactions mechanistic insights.

1. Solomon E.I., Sundaram U.M., Machonkin T.E.. Chem. Rev., 96, 2563-2605 (1996)

Toxic metal interactions with zinc f ingers

Anne M. Rich, Aust in D. Schenk, Dean E. Wilcox

Department o f Chemistry, Dartmouth College, Burke Laboratory, Hanover. New Hampshire, 03755~ U.S.A. (e-mai l . anne . r ich@dar tmouth .edu)

Fhe metal binding properties of zinc finger peptides and Zn-binding domains of transcription factors have been characterised with absorption and CD spectroscopy and isothermal titration calorimetry _ ~ , (ITC). Studies of the DNA-binding domain of glucocorticoid receptor (GR), which binds ~:/~ _ , .:: two Zn(II) ions with C4 ligation, and a peptide corresponding to the second Zn-binding ":~ !~,". site (GR-2), have indicated the relative metal affinities and quantified the dissociation i ~ constants, which for GR-2 are: Ni 2+ (4.8 4- 0.5 x 10 -4) < Co 2+ (3.5 + 0.4 x 104) < Zn z+ (4.1 ~!! ± 0.8 x 10 -6) < Pb 2+ (1.2 + 0.7 x 10 "7) ~ Cd 2+ (2.1 4- 0.7 x 10"7). Analogous studies with g ~ - ' ~ ~ : %, peptides containing the classical (C2H2) zinc fingers of the DNA-binding domain of Spl, ~'~ .... reveal higher affinities. ITC has been used to quantify the thermodynamics associated ~L,_ with metal binding, including enthalpic and entropic contributions and the number of protons released upon metal ligation. These results indicate the relative metal and protein contributions to the stability of zinc fmgers and zinc-binding domains, and have implications tbr the effects of toxic metals on DNA binding by GR, Sp 1 and other transcription factors that require Zn(II) for stabilization of a protein structure that is competent for recognition and binding to DNA..

The Dartmouth Superfund Basic Research Grant is acknowledged for financial support.

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400 Journal of Inorganic Biochemistry 86 (2001)

Axial l igand modulation of a low-lying excited state in the thermus CUA domain

John H. Richards a, Claudio O. Femfindez b, Julia A. Cricco c, Claire E. Slutter a, Harry B. Gray a, Alejandro J. Vila c, Igor Gromov d, Boris Epel d, Daniella Goldfarb a, Israel Pecht e

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 (email: [email protected])

b LANAIS RMN-300 (UBA-CONICET), Junin 956, CII3AAD Buenos Aires, Argentina c Biophysics Section, University of Rosario, Suipacha 531, S20021LRKRosario, Argentina d Departments of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel, 76100. e Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, 76100.

The Cu A center is the initial electron acceptor in cytochrome c oxidases and nitrous oxide reductases. The weak axial ligands of this center have been proposed to mediate Cu-S core distortions that are vital in modulating the reduction potential of the center [Gamelin, et al. J. Am. Chem. Soc., 120, 5246-5263 (1998).] An EPR characterization of the axial ligand mutants of Thermus CUA, MI60Q and M160E, has shown that these altered axial interactions shift more electron spin density from the ligands to the Cu centers. The paramagnetic NMR spectra of CUA show anomalous temperature dependences of the ~-CH2 Cys protons, a feature attributed to a population of a low-lying, thermally accessible, excited state. Analysis of this temperature dependence in the M160Q mutant reveals that the excited state has shifted to higher energy compared to the wild-type protein. Moreover, the axial mutation only affects resonances assigned to residues that coordinate Cub not Cu2. Thus, axial ligand perturbations in the Thermus CUA site leads to subtle structural changes and retention of the au* ground state; but, significantly, alters the energy gap between the ground state and low-lying excited states. Because this excited state is populated at physiologically relevant temperatures, we propose that electron injection occurs via the CUA excited state. This control mechanism yields a low reorganization energy and provides a larger range of regulatory possibilities in the mitochondrial oxidases than earlier proposals that rely on Cu-S core deformations and switching of the ground state molecular orbitals.

Alkyl nitrite (RONO) and -thionitrite (RSNO) additions to group 8 metal loporphyrins to give nitrosyl alkoxides and -thiolates

George B. Richter-Addo. Jonghyuk Lee Department of Chemistry and Biochemistry,

Norman, Oklahoma, 73019, USA (e-mail: University of Oklahoma, 620 Parrington Oval,

grich teraddo @o u. edu)

The role of metal ions in the decomposition of biologically-active alkyl nitrites (RONO) and alkyl thionitrites (RSNO) continues to attract attention in the chemistry and biochemistry of nitric oxide. We have previously reported that the group 8 metalloporphyrins react with RONO or RSNO compounds to give (por)M(NO)(OR) or (por)M(NO)(SR) derivatives, respectively.I In the case of iron, the reaction of RSNO with ferrous (TPP)Fe(THF)2 produces the five-coordinate (TPP)Fe(NO) as the fmal product.

We have extended the formal addition reactions to include a wide variety of RSNO compounds (a representative molecular structure is shown in the Figure), and we have also generated the (por)M(NO)(SR) products by other routes as well. 2 For example, we have prepared the bimetallic [(por)Os(NO)](~t-dithiolate)[Ru(NO)(por)] compound by the reaction ~. ,~ of (por)Os(NO)(SCH2CH2SH) with t-BuONO and (por)Ru(CO). Various aspects of the L.]~ reactions to produce the (por)M(NO)(SR) products will be discussed, as will the solid-state crystal structures of the (por)M(NO)X family of complexes that reveal unusual axial distortions of the ligands.

. C , r ,

1. Richter-Addo G. B., Accounts of Chemical Research, 32, 529-536 (1999). 2. Lee J., Yi G.-B., Powell D. R., Khan M. A., Richter-Addo G. B., Canadian Journal of Chemistry, in press.

The National Science Foundation and the National Institutes of Health of the USA is acknowledged for financial support.