abstracts poster presentations (e)

Journal of lnorganic Biochemistry 86 (2001) 161

Syntheses and metal ion binding strengths of desferrioxamine B model dihydroxamic acids

P r t e r B u g l y 6 a, E t e lka Fa rkas a, l~va A n n a E n y e d y a, V e r o n i k a Gerle i a and M. A m r l i a San tos b

ODepartment of Inorganic and Analytical Chemistry, University o f Debrecen, H-4010 Debrecen, Hungary (e-mail: [email protected])

bCentro de Quimica Estrutural, Complexo I, lnstituto Superior TOcnico, 1049-O01 Lisboa, Portugal

New dihydroxamic acids (a-c) with different position of the peptide bond between the two functional groups (a-b) or with shorter chain (c) have been synthetised in order to explore the role of the structural constituents of desferrioxamine B (DFO) in metal ion binding, where DFO is a naturally occuring tris-hydroxamate type siderophore.

The appropriate amino carboxylic acids were reacted with glutaric or succinic anhydride to form the intermediate peptides and these were converted into the ligands (a-c) using N-methyl-hydroxylamine.

CH3 O O n m ligand ..IN. ~ . ~ ~ ~ ~ ..OH 1 4 a

" 0 T~, n/~ N~ m]~ N 2 3 b O CH 3 2 2 c

The results of pH-potentiometric and UV-VIS spectroscopic equilibrium measurements with V(V), Mo(VI) and Fe(III) indicate that in all these systems studied ligand a with 2-5 arrangement of the peptide bond between the hydroxamates forms complexes with significantly higher stability than ligands b or c, This finding strongly supports that desferrioxamine B has highly preorganised structure and the position of the peptide bonds plays a crucial role in its metal binding capabilities.

The Hungarian Scientific Research Fund (OTKA T034674), the Jfinos B61yai Research Grant and the Hungarian- Portuguese Intergovemmental S & T Co-operation Programme for 2000-2001 are acknowledged for financial support.

Cyanide binding to KatG and KatG(S315T): A kinetic model of superoxide binding

Vanja M. Bulatovic, Nancy L. Wengenack, and Frank M. Rusnak Section of Hematology Research and the Department o f Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, (e-mail: Bulatovic. [email protected])

Isoniazid, an antibiotics used to treat TB, is a prodrug activated by the Mycobacterium tuberculosis hemoprotein KatG, a catalase-peroxidase that also exhibits Mn>-dependent peroxidase and cytochrome P450-1ike monooxygenase activities. A common mutation causing isoniazid-resistant Mtb strains is

i " CN KatG(S315T).Wild type (WT) Mtb KatG and KatG(S315T) are both competent ~ . ) x | o catalase-peroxidase enzymes, but KatG(S315T) is less efficient at converting INH to - - ~ isonicotinic acid via a mechanism involving superoxide as oxidant. The hypothesis that L ~" I KatG(S315T) has a decreased superoxide reactivity compared to WT KatG due to ~'/ j"?r-cH,- cH2- decreased accessibility of the active site Fe ion can be investigated by following the L _ 2 binding of a C N - as a mimic of superoxide. CN-- binds KatG, resulting in an absorbance increase at 426nm. The rate of C N - binding in the presence/absence of isoniazid was measured in order to determine whether isoniazid binding affects the rate of ligand binding. The data indicate that C N - binds both KatG and KatG(S315T) rapidly. Progress curves were best fit by three exponential terms yielding three pseudo first order binding constants, representing fast (k0, intermediate (k2), and slow (k3) rates of binding. C N - binds 5-coordinate high-spin KatG rapidly (kl) and 6-coordinate low-spin KatG at an intermediate rate (kz). The slow rate (k3) is hypothesized to represent a small amount of WT enzyme which reluctantly binds CN- . The presence of isoniazid increased the fraction of WT enzyme that binds CN~ rapidly (k~), with concomitant reduction in the fraction that bound 1NH at intermediate and slow rates, converting the WT enzyme to a homogenous species accessible to ligand binding. In contrast to WT KatG, isoniazid fails to cause a transition of KatG(S315), suggesting that the $315T mutation alters the reactivity of the iron with small ligands like cyanide, and by analogy, superoxide, leading to a decreased ability to oxidize isoniazid.

162 Journal of lnorganic Biochemistry 86 (2001)

M a n g a n e s e transferr in

Kerry E. Bunyan a'b, Benjamin J. Tura b, Ian Harvey c, Shailja Bihari a, David J. Harrsion b and Peter J. Sadler"

Department of Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3J J, UK (e-mail: pjs28@holyrood, ed.ac.uk) b Department of Pathology, University Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK c CLRCDaresbury Laboratory, Warrington WA4 4AD, UK

Reactive oxygen species are continuously produced during cell metabolism. In mitochondria, a major antioxidant is manganese superoxide dismutase. But how is manganese transported and delivered to cells? - probably by the major iron transport protein, serum transferrin.1 Transferrin is an 80 kDa single-chain bilobal glycoprotein with two similar metal binding sites. Human apotransferrin (apo-hTF) binds weakly to Mn(II), but more strongly to the highly acidic ion Mn(lll). Reaction of apo-hTF with Mn(II) in air leads to only a very slow uptake of Mn(III) (weeks at 310 K). This reaction appears to be catalysed by the serum oxidant protein caeruloplasmin (days). In contrast, Mn insertion via reduction of MnO4- by Hepes buffer at pH 7.4 leads to a more rapid binding of Mn(III) (hours). X-ray spectroscopy (EXAFS and XANES) have been used to investigate the oxidation state of Mn bound to hTF, and to compare the coordination spheres of Mn in mono- and di-Mn transferrin. We are investigating the potential role of Mn-transferrin in the cellular uptake of Mn, using a model for liver injury involving rescue of primary murine hepatocytes from mitochondrial-mediated apoptosis induced by interferon-?,.

1. Sun, H., Li, H. and Sadler, P.J., Chem. Rev., 99, 2817-2842 (1999)

We thank the BBSRC, Wellcome Trust, MRC and EPSRC for their support for this work.

Synthes i s of a N e w F l u o r e n e - B a s e d Ligand System to Enforce a Cs-Symmetr ic C o o r d i n a t i o n G e o m e t r y in C a r b o x y l a t e - B r i d g e d C o m p l e x e s

Dirk Burdinski, Karen Cheng, Stephen J. Lippard Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (email: contact@dirk-burdinski, de)

Methanotrophic bacteria catalyze the selective oxidation of methane to methanol by a multicomponent enzyme system. Its hydroxylase component (sMMOH) contains a carboxylate-bridged non-heine diiron center in the active site. During catalysis, a high-valent FeW2(~t-O)2 species is formed that formally inserts one of the two bridging oxygen atoms into an aliphatic C-H bond] The oxidation of a dangling benzylamine functionality to benzaldehyde has recently been achieved with a Ci-symmetric diiron model complex bearing four carboxylate and two anti disposed nitrogen donor groups. 2 The respective N-donor atoms in sMMOH are arranged in a Cs-symmetric, syn fashion, however. The Cs ligand arrangement may be important for the selective activation of one of the two oxo bridges for substrate oxidation. The ligand system 1 was therefore designed and synthesized in eight steps to enforce such a C~-symmetric coordination geometry for use in the next generation of functional model complexes.

l. Gherman, B. F., Dunietz, B. D., Whittington, D. A., Lippard, S. J., and Friesner, R. A., J. Am. Chem. Soc., 123, 3836-3837 (2001)

2. Lee, D. and Lippard, S. J., J. Am. Chem. Soc., 123,4611-4612 (2001)

I I

I 1 1

Supported by NSF and NIGMS. D. B. thanks the Deutsche Forschungsgemeinschaft for an Emmy-Noether Fellowship.

Journal of Inorganic Biochemistry 86 (2001) 163

Photochemical properties of the [Ni-Fe] center in hydrogenases: role of the terminal sulfur l igands of the Ni ion in the photoinduced proton transfer

B6n6dicte Burlat, Val6rie Belle, Marcel Asso, E. Claude Hatchikian, Christine Cavazza, Marc Rousset, Patrick Bertrand, Bruno Guigliarelli

UnitO de Bio~nergOtique et Ing~nierie des ProtOines, CNRS, 31 Chemin J. Aiguier, 13402 Marseille cedex 20, FRANCE.

Hydrogenases are enzymes which catalyze the reversible oxidation of molecular hydrogen. An important class of these enzymes contains a catalytic site composed of a dinuclear [Ni-Fe] cluster. Upon activation under hydrogen atmosphere, the NiFe hydrogenases give the well-known Ni-C species that is considered as an intermediate of the catalytic cycle. This species is light-sensitive, and can be converted at cryogenic temperature into various Ni-L species. These conversions are considered to correspond to photodissociations of hydrogenated species from the [Ni-Fe] center. Moreover, we have shown that they are accompanied by the complete cancellation of the exchange interaction between the [Ni-Fe] center and the proximal [4Fe-4S] ~+ of the enzyme[ 1 ]. These conversions are fully reversible at temperature higher than 10OK.

In order to progress in the understanding of the Ni-C species structure, we have performed a detailed EPR study of the various photoconversion and recombination processes in the NiFe hydrogenase from Desulfovibrio fructosovorans, and in the NiFeSe enzyme from Desulfomicrobium baculatum in which one of the terminal Cysteine ligand of the Ni ion is replaced by a seleno-cysteine. Wavelength dependence of the photoconversions and of their kinetic parameters showed that the modifications of the [Ni-Fe] center and the cancellation of the exchange coupling originate from the same photoevent. Moreover, the activation energies of the recombination processes were determined in both hydrogenases and show a strong increase from NiFeSe to NiFe enzymes which likely reflects the difference of acidity between -Sell and - SH groups. These results indicates that only the coordination sphere of the Ni ion is involved in the studied photoconversions and can be interpreted in terms of a photoinduced proton transfer occurring between the terminal sulfur ligands of the Ni ion of the [Ni-Fe] cluster. The amino acid possibly involved in this proton transfer are discussed.

1. Dole, F., Medina, M., More, C., Cammack, R., Bertrand, P., Guigliarelli, B. (1996) Biochemistry 35, 16399-16406.

Cobalt /Zinc as structural elements of bacterial adenylate kinase

Sergey A.Bursakov", Olga Yu.Gavel", Giulia Di Rocco a, Jorge Lampreia a, Valery L.Shnyrov b, Graham N.George ~, Juan J.Calvete d, Jose J.G.Moura a , Isabel Moura a

"Dept. Quimica, Faculdade de Ci6ncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte de Caparica, PORTUGAL (e-mail. serger(~dq.fct, unl.pt)

~Dept. Bioquimica y Biologia Molecular Universidad de Salamanca, Plaza de los Doctores de la Reina, s/n 37007 Salamanca, SPAIN

CStanford Synchrotron Radiation Laboratory, Stanford University SLAC, P.O. Box 4963, Stanford, California 94309, U.S.A.

~Instituto de Investigaciones BiomOdicas, C.S.I.C., Valencia, SPAIN

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their cellular constant level, necessary for energetic metabolism and nucleic acid synthesis. Gram-positive organisms harbors a structural Zn 2+ bound to 3 or 4 Cys residues in the structural motif in contrast with AK from Gram-positive bacteria, which are usually devoid of metal ion. We present here AKs from Gram- positive bacteria Desulfovibrio desulfuricans ATCC 27774 (Dd) and D. gigas (Dg) containing cobalt and zinc as structural element. AK(s) from this sulfate-reducing bacteria are monomers (24.9 kDa and 24.7 kDa for Dd and Dg proteins). The electronic absorption spectra display charge-transfer bands consistent with at least three sulfurs coordinated to cobalt, and as expected for a distorted tetrahedral cobalt geometry, d-d bands at 620, 648, and 688 nm. This geometry is supported by the observation of a high-spin Co 2. (S = 3/2) EPR signal at g -~ 7.27 exhibiting hyperfine splitting 309.7 G (A = 44.2 G) due to the nucleus of 59Co (I = 7/2). EXAFS data reveal that either cobalt or zinc binds endogenously at presumably equivalent metal binding sites and is tetrahedrally coordinated to three sulfur and one oxygen/nitrogen. The sequence of the D.gigas enzyme confirm the presence of the three cysteines attributed to the metal binding site. Metal substitution techniques as well as thryptophan fluorescence study enable to attribute this motif as a structural metal binding site derivative.

PRAXIS XXI/SFRH/BPD/3518/2000/BD/13775/97 is acknowledged for financial support.

164 Journal of Inorganic Biochemistry 86 (2001)

Zinc and iron complexes with bis(pyrazol-l-yl)acetato ligands: models for zinc and mononuclear non-heme iron enzymes

N. B u r z l a f f , A. B e c k , Department o f Chemistry, University o f Konstanz, Universitiitsstrafle 10, Fach M728, D-78457 Konstanz, Germany (e-mail: n i c o l a i @ c h e m i e . u n i - k o n s t a n z . d e )

There has been a significant progress in the structural characterisation of mononuclear non-heme iron oxidases such as IPNS, DAOCS or CAS over the last ten years. Metal complexes ofbis(pyrazol-l-yl)acetic acids can mimic their so called "2-His-l-carboxylate facial triad 'a as well as the active sites of zinc enzymes. Therefore, several bis(pyrazol-l-yl)acetic acids such as bis(pyrazol-l-yl)acetic acid (bpzaH) (1), bis(3,5-dimethylpyrazol-l-yl)acetic acid (bdmpzaH) (2) and bis(3,5-di-tert-butylpyrazol-l-yl)acetic acid (bdtbpzaH) (3) have been synthesised. 24 The sterically more hindered ligand 3 ligates only once to Zn(II) resulting in the complexes [(bdtbpza)ZnCl] (4) and [(bdtbpza)ZnOAc] (5) as structural model complexes for the active sites of zinc enzymes such as thermolysin or carboxypeptidase. Reaction of 3 with FeCI2 led to a dimer [(bdtbpza)FeC1]z (6). The Fe(III) complexes [NEt4][(bpza)FeC13] (7) and [NEt4][(bdmpza)FeC13] (8) are reported too. Complexes 6, 7 and 8 might be future precursors for structural model complexes of mononuclear non-heme iron oxidases and oxygenases such as IPNS, DAOCS or CAS.

1. Hegg E.L., Que Jr. L., Eur. J. Biochem., 250, 625 - 629 (1997). 2. Otero A., Ferngndez-Baeza J., Tejeda J., Antifiolo A., Carrillo-Hermosilla F., Diez-Barra E., Lara-S~nchez A.,

Fem~ndez-L6pez M., Lanfranchi M., Pellinghelli M.A., J. Chem. Soc., Dalton Trans., 3537 - 3539 (1999). 3. Beck A., Weibert B., BurzlaffN., Eur. J. Inorg. Chem., 521 - 527 (2001). 4. BurzlaffN., Hegelmann I., Weibert B., J. Organomet. Chem., 626, 16 - 23 (2001).

Protein film voltammetry of a respiratory nitrate reductase: evidence of complex electrochemical modulations of enzyme activity

Ju lea N. Bu t t a, Le e J. A n d e r s o n a , a n d D a v i d J. R i c h a r d s o n b

a School of Chemical Sciences, Centre for Metalloprotein Spectroscopy and Biology, University o f East Anglia, Norwich, NR4 7TJ, UK (e-mail. j [email protected])

b School o f Biological Sciences, Centre for Metalloprotein Spectroscopy and Biology, University o f East Anglia, Norwich, NR 4 7 T J, UK

Paracoccus pantotrophus develops a respiratory chain terminated by a membrane bound quinol:nitrate oxidoreductase, NarGHI, when grown anaerobically with nitrate as the respiratory oxidant.l The dimer, NarGH, can be liberated from P. pantotrophus membranes as a functional nitrate reductase which binds four [Fe-S] clusters in addition to a molybdenum-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) containing catalytic site. 2 NarGH self-assembles as a functional film on graphite and gold electrodes) Within the film electrons exchange directly between the electrode and the enzyme which allows the enzyme's catalytic performance to be visualised over a wide potential range. Most interestingly the shape of the catalytic response changes as the substrate (nitrate,chlorate or proton) concentration is changed. The complex pattern of electrochemical and chemical modulations of catalytic activity exhibited by NarGH allow us to suggest models that begin to explain the enzyme's catalytic performance in terms of chemistry and electrochemistry occurring at the [Fe-S] clusters or Mo-bis-MGD centre of the enzyme.

1. Richardson, D. J., Microbiol. 146, 551-571 (2000) 2. Ballard, A. L. and Ferguson, S. J. , Eur. J. Biochem 174, 207-212 (1988) 3. Anderson L. J., Richardson D.J. and Butt J.N., Faraday Discuss., 116. 155-169 (2000)

We acknowledge The Wellcome Trust (Grant No 050709) for financial support.


The reaction of Desulfovibrio vulgaris rubredoxin oxidoreductase (desulfoferrodoxin) with superoxide

D i a n e E. Cabe l l i b, J o s e p h P. E m e r s o n , a Er ic D. Coul te r , a D o n a l d M. Kur tz , Jr. a

Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, U.S.A. bChemistry Department Brookhaven National Laboratory Upton, NY 11973 (email. [email protected])

Rubredoxin oxidoreductase (Rbo) from D. vulgaris functions as a superoxide reductase (SOR), catalyzing reduction of 02- to H202. We have characterized a 600-nm absorbing intermediate during the reaction of superoxide with the ferrous Center II (RbOpmk). l This intermediate is formulated as a ferric-(hydro)peroxo species, which decays to a six- coordinate carboxylate-ligated ferric Rbogray with loss of H202) Additional pulse radiolysis studies now show that the first diffusion- controlled phase, consisting of oxidation of the ferrous Center II by 02-, is independent of pH and shows no D20 effect, whereas, the rate of the second phase, consisting of first-order decay of the 600-nm intermediate, increases with decreasing pH and shows a substantial D20 effect. These results, together with mutational studies, show that protonation of the

sc?~ Rbo=i~.

~,~a C~rH~,

H 1 F'hi

8C~,s 600-rim ~nterrnodiale

GkJ47~

40 t ,~

O~/C -G'~ 47 ~b%'-u,

Rbogra Y (647 rim)

ferric-peroxo intermediate is an essential feature of the SOR mechanism catalyzed by Rbo and related SORs.

1. Coulter E. D., Emerson, J. P., Kurtz, D. M., Jr. and Cabelli, D. E. J. Am. Chem. Soc. 122, 11555-11556 (2000)

This research was supported by NIH grant GM40388 and contract DE-AC02-98CH 109916 with the U.S. Department of Energy

Nitrous oxide reductase (N2OR) from Pseudomonas nautica 617

in6s Cabr i to a, A l i ce S. Pere i ra a, Ped ro Tava re s a, S t6phane B e s s o n a, Car los B r o n d i n o a'b, Br ian H o f f m a n b, K . B r o w n c, M . T e g o n i c, C . C a m b i l l a u c, Jos6 J. G. M o u r a ~ and Isabel M o u r a ~

"Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de CiYncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal, bDepartment of Chemistry, Nortwestern University, Evanston I 60208 - 3113, USA;CArchitecture et Fonction des MacromolOcules Biologiques, URA 9039, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex, France (e-mail: i m g @ d q . f c t . u n l . p t )

N2OR reduces nitrous oxide into dinitrogen in many denitrifying bacteria. N2OR is a homodimer of q 2 0 k D a containing two copper clusters per monomer: a binuclear, CuA and a tetranuclear, CuZ. Under aerobic conditions two form of N2OR were purified, which were proven to be redox intermediates between fully oxidized and fully reduced N2OR (different oxidation states of CuA since Cuz didn't change its oxidation state). CUA is a mixed-valence [Cu+2Cu ÷l] cluster with the coppers bounded by two Cys Sy and two His, a Cys St, and a Trp binding externally. Oxidized CUA exhibits a typical 7-line EPR spectrum and absorptions at 480, 540 and -800nm; and a EPR-silent reduced state. Cuz comprise 4 copper arranged in a distorted tetrahedron with seven His ligands. Coppers I, II, III are ligated by two His, whereas only one His ligates CuIV. A S atom is a bridging ligand between the copper atoms and an oxygen specie binds CuIV and CuI, though its nature is not yet known. Cuz exhibits a 4-1ine EPR and a strong absorption at -640 rim. To gain more information on the nature of the ligands and on the redox states of the copper ions, we performed 35 GHz CW proton and nitrogen ENDOR studies. Results will be discussed in terms of oxidation states of the copper ions, spin delocalization and nature of the ligands. 1 - Prud6ncio M, A.S.Pereira, P.Tavares, S.Besson, I.Cabrito, K.Brown, B.Samyn, B.Devreese, J.Van Beeumen, F.Rusnak, G.Fauque, J.J.G.Moura, M.Tegoni, C.Cambillau and I.Moura, Biochemistry 39, 3899-3907 (2000) 2 - K.Brown, M.Prudfincio, A.S.Pereira, S.Besson, J.J.G.Moura, I.Moura, M.Tegoni and C.Cambillau, Nature Sstructure Biology 7, 191-195 (2000) 3 - K.Brown, K.D.Carugo, T.Haltia, I.Cabrito, M.Sarate, J.J.G.Moura, I.Moura, M.Tegoni and C.Cambillau, J. Biol. Chem. 275:41133-41136 (2000) We thank PRAXIS and BIOTEC for financial support.


Biphosphinic palladacycle complexes as antitumoral agents

Antonio C.F.Caires a, Simone D.Rodrigues a, Luiz R.R.G.Travassos b, Elaine G. Rodrigues b ~CIIB, Universidade de Mogi das Cruzes, CP.'411, 08701-970,Mogi das Cruzes-SP, BRAZIL, (e- mail caires(~umc.br) bDepartamento de Cidncias Biom~dicas, UNIFESP-S6o Paulo,BRAZIL

In a previous paper we report that palladacycle compounds having biphosphinic ligands are potential antitumoral agents L. In the attempt of propose mechanisms and structural correlations concerning the biological interactions of this class of organometallic complexes, new compounds have been synthesized and tested as antitumoral agents. Thus, from palladacycle compounds generated by enantiomers R (+) and S(-) of N,N-dimethyl- 1-phenethylamine 2 and functionalized alkynes, several complexes having biphosphinic ligands have been obtained. The neutral or ionic, mono or binuclear complexes synthesized were characterized by elemental analyses, IR spectroscopy and ~H and 3~p NMR techniques. The series of complexes were screened for cytotoxicity against the cell lines B16F10 of murine melanoma and NIH3T3 of fibroblasts. Besides important correlations involving structure of complexes and biological activity, it was found that the cyclopalladated compounds having the ligand 1,2-bis(diphenylphosphine)ethane, dppe, are effective antitumoral agents. Measurements of plasmatic membrane potential shows a selectivity of the complexes containing dppe ligand in specific concentration ranges. In some defined concentration of cyclopalladated compounds the normal cells have a increase in its metabolism while the tumoral cells die.

1-Caires,A.C.F.; Almeida,E.T.; Mauro,A.E.; Hemerly,J.P.; Valentini,S.R.; Quimica Nova, 22(3), 329- (1999) 2-Ryabov,A.D.; Kazankov,G.M.; Kurzeev,S.A.; Samuleev,P.V.; Polyakov, V.A.; Inorg. Chim.Acta, Protein,280,57(1998)

The Funda~o de Amparo/1 Pesquisa do Estado de S~o Paulo (FAPESP) is acknowledge for financial support

Synthesis and characterization of nickel(II) and manganese(III) complexes of tetraazaannulene and encapsulation in porous Vycor glass (PVG)

Jos6 Mauricio A. Caiut, Shirley Nakagaki, Geraldo Roberto Friedermann, Rodrigo Arafijo Franga and Aldo Jos6 Gorgatti Zarbin Department of Chemistry, Federal University of Parand, Jardim das AmOricas s/n, zip. 81531-

990, P.O. 19081, Curitiba, Brazil. (e-mail: [email protected], br

The metal tetraazacyclotetradecine complexes (tetraazaanulene, e.g. NiTMTAA) have attracted much recent attention because their common characteristics with metalloporphyrins and possibility to use in "-- . .~ ' - .~/ biomimmetic systems. However, these complexes exhibit framework flexibility due to their smaller conjugation if compared with the porphyrins that can allow the central metal ~ , ~ N',,N N . ~ / ~ to easily change oxidation states ~. The facility to introduce variations on the structure of i2{ tetraaza also is an attractive property of these compounds for a model to metalloenzyme - v ,~ , ' N / \N" ~ / (Factor F430 and cytochrome P450) 2. The PVG is an interesting support to catalysts like metallocomplexes because, the surface of porous has silanol groups that collaborate to immobilization of the complexes and the solid presents good stability in the catalytic study conditions. The immobilization of NiTMTAA and MnTMTAA into the PVG was accomplished successfully by UV-Vis. Analysis of electronic paramagnetic resonance showed change oxidation state of manganese tetraazacyclotetradecine ( Mnm--~Mn H ) during the process. The use of the MTMTAA-PVG compounds in the oxidation and reduction reactions as model of cytochrome P450 and Factor F-430, respectively is in progress and has shown good perspectives.

1- Arai T., Kashitani K., Kondo H., Sakaki S., The Chemical Society of Japan, 67, 705-709 (1994). 2- Eilmes J.Polyhedron, t 1, n o 5,581-584 (1992)

Acknowledgment: UFPR, CAPES, CNPq, LACTEC, FUNPAR, PADCT


About arginase

Evis Cama Depar tmen t o f Chemis t ry Univers i ty o f Pennsy lvan ia Phi ladelphia , PA USA

Arginase is a 105 kDa homotrimer containing a binuclear manganese cluster in each monomer that catalyzes the hydrolysis of arginine to L-ornithine and urea. The crystal structure of liver arginase, also designated as type I arginase, has been determined to 2.1A resolution. Structure-based designing of the boronic acid analogue of L-arginine, 2(S)- amino-6-boronohexanoic acid (ABH), brought about the synthesis of this slow binding competitive inhibitor of arginase. The structure of the complex reveals the way in which ABH yields a tetrahedral boronate anion, mimicking the tetrahedral transition state of L-arginine. This transition state does not occur in NO synthase, explaining why ABH does not inhibit NO synthase. The intermediate in the NOS hydrolysis pathway of L-arginine to L-citrulline and NO, N~°-hydroxy-L - arginine (NOHA), is a modest competitor of arginase. The structure of arginase-NOHA complex provided further insight into the catalysis of arginase as well as a mechanistic link between arginase and NOS. Crystal structures of complexes of arginase with modified versions of these two inhibitors provide further insight into the role played by each of the functional groups of these compounds.

Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system

Chantal Capeillere-Blandin, Frangoise Auchbre, Gildas Bertho, Isabelle Artaud, Jean Pierre Girault Labora to i re de Chimie et B ioeh imie pharmaco log iques et tox ico logiques (UMR 8601CNRS) UniversitE Paris 5, 45 rue des Saints P~res 75270 Paris Cedex 06, FRANCE. (E.mai l : chantal, b land in@biomed ica le , univ-par is5 . f r )

Haem-enzyme myeloperoxidase (MPO) catalyzes the reaction of chloride ion with hydrogen peroxide (H202) to

generate the potent microbicidal agent hypochlorous acid (HOC1). The spectrophotometric study of the reaction of the MPO / H202 / chloride system with NADPH and NMNH showed that the reaction products were not the corresponding

oxidized nucleotides and that modifications would take place on the nicotmamide part of the molecule.l To obtain more precise information on the structural modifications and mechanism of the reaction the NADPH and NMNH derived products were purified and isolated by reverse phase HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35C1 and 3Vc1, providing evidence that the NADPH and NMNH derived products were monochlorinated. Moreover, calculated masses revealed the 1:1 addition of HOC1 to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCI addition occurred at the C5 = C6 carbon double bond of the nicotinamide ring, thus leading to a chlorohydrin.

1. Auchbre, F. and Capeill6re-Blandin, C., Biochem. J., 343,603-613 (1999)


Modeling the active site of cytochrome oxidase: synthesis and characterization of a cross-linked histidine-phenol

Jenny A. Cappuccio a, Idelisa Ayala b, Gregory I. Elliott a, Istvan Szundi a, James Lewis a, Joseph P. Konopelski a, Bridgette A. Barry b, and O16f. Einarsdottir a

aDepartment of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High St., Santa Cruz, CA 95064, USA (e-mail: jenny@chemistry. UCSC. edu)

bDepartment of Biochemistry, Molecular Biology, and Biophysics, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108, USA.

A cross-linked histidine-phenol compound, a chemical analog of the active site of cytochrome c oxidase, was synthesized. The structure was verified by IR, IH and 13C NMR, mass spectrometry, and by single crystal X-ray analysis. Spectrometric titrations indicated that the pKa of the phenolic proton on the model cross-linked His-phenol (8.34) was lower than the pKa of tyrosine (10.4) or of p-cresol (10.2). This decrease in pKa is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time resolved optical absorption difference spectra of the His-phenol compound generated by excitation at 266 nm showed a unique absorption band at 500 nm, significantly shifted from the 400 nm maximum observed for the UV-generated tp'rosyl radical. EPR spectra of the His-phenol model compound, obtained after UV photolysis, confirmed the formation of a paramagnetic species at low temperature. Simulation of the EPR lineshape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground state FT-IR spectrum of the His-phenol model compound showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. Taken together, these results indicate that the unpaired spin density in the radical form of the His-phenol compound is delocalized primarily on the phenoxyl ring and that optical spectroscopy can detect the unique structural properties of the cross-link.

Reactive intermediates in binuclear non-heme iron catalyzed oxygen atom transfer reactions

John P. Caradonna a, Ann Marie Spuches b, Cory Rogge b, Trina Foster a ~Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston MA USA (e-mail: [email protected]) bDepartment of Chemistry, Yale University, 225 Prospect Street, New Haven CT06520

02215,

The ability of a series of binuclear non-heme iron ferrous complexes (la-e) to catalyze the (i) hydroxylation of arenes and alkanes (alcohol:ketone > 50:1), (ii) epoxidation of olefms, and (iii) oxidation of sulfides, in the presence of a variety of oxygen atom donor molecules will be discussed. The identification and preliminary characterization of kinetically competent intermediate species, (2a-e), generated by the stoichiometric reaction of the diferrous complexes with oxygen atom donor molecules at -80 °C will be presented. Production of 2a-e causes disruption of the integer spin EPR signal characteristic of the ferromagnetically coupled diferrous cores of la-e and leads to the generation of EPR silent species. The low temperature addition of either adamantane or methylphenylsulfide to solutions of 2b results in the clean decay of the iron-based intermediate and yields 2-adamantanol and methylphenylsulfoxide, respectively. The exclusive production of 2-adamantanol at low temperatures contrasts the 30/2 ° C-H oxidation ratio of 4:1 observed in room temperature reactions, suggesting a change in thermodynamic vs. kinetic control of the oxidation pathway. In the absence of substrates, 2a-c quantitatively decay into diamagnetic g-oxo diferric species which are inert as an oxygen atom transfer catalysts. Comparable studies using the mixed valent binuclear iron complexes, 3a-e, indicate the presence of analogous reactive intermediates, 4a-c, that are also competent oxygen atom transfer species. The abilities of both the diferrous and mixed valent core oxidation states to induce heterolytic decomposition of phenylperacetic acid, catalyze oxygen atom transfer reactions, and perform one and two electron oxidation reactions will be discussed in terms of the proposed mechanisms for the metalloenzymes methane monooxygenase (MMO) and ribonucleotide reductase R2 (RNR). Insights into the natures of the model-based reactive intermediates and their comparisons to the putative intermediates of the enzyme systems will be presented.


Synthesis and biological action studies of metal complexes containing squarate-substituted cyclophosphazatriene as ligand

G e o r g e B. C a r a m a n a, A n c a B e n e b, V e r o n i c a M a r i n a, F l o r i n a D u m i t r u c, C o r n e l i a G u r a n c

a Institute of Organic Chemistry "C.D. Nenitescu", Spl.Independentei 202-204, 70000, Bucharest, Romania, (e-mail. [email protected]) b "Cantacuzino" Institute, Spl. Independentei 103, Bucharest, Romania c Department of lnorganic Chemistry, UniversityPolitehnica of Bucharest, Polizu 1, 78126 Bucharest, Romania

A large number of cyclophosphazatriene derivatives present biological uses ~. In our previous works we presented data about the antifungal activity against Aspergillus and Candida spp., of some d metal complexes of 1,3,5-tris(8- hydroxyquinolino)-trichloro-cyclophosphazatriene and of dichloro-tetramorpholino-cyclophosphazatriene 2'3. Here we present the synthesis and characterization of the Fe 3+, Cr 3+ complexes with cyclophosphazene squarate- substituted derivatives as well as biological action studies on these new compounds. The cyclophosphazene squarate-substituted derivatives were successfully prepared in well established conditions (solvent, reaction time, temperature, molar ratio) starting from hexachlorocyclotriphosphazene and squaric acid ( 3,4-dihydroxy-3- cyclobutene- 1,2-dione). The obtained complexes were assayed as inhibitors against several widespread fungi, such as Aspergillus, Actinomicetae, Blastomices. Sporotrix and Candida spp., evidencing interesting activity for some of them.

1. Allcock H. R., Dodge J., Manners J. A., Riding G., J. Am. Chem. Soc., 113, 9596-9603, (1991); M. Witt, H. Roesky, Chem. Rev., 94, 1163-1181, (1994)

2. Guran C., Barboiu M., Jitaru I., Supuran C.T., Metal Based Drugs, 3,233, (1995) 3. Guran C., Barboiu M., Supuran C. T., Diaconescu P., Iluc V., Metal Based Drugs,, 5(5), 287, (1998)

Characterization of Cox l l , a putative mitochondrial copper chaperone

H. S. Carr, D. R. Winge University of Utah Health Sciences Center, Salt Lake City, UT84132, USA (e-mail:heather. carr@hs c. utah. edu)

Cytochrome c oxidase (CCO) contains several cofactors which are necessary for catalytic activity, including two hemes a, a magnesium ion, a zinc ion, and 3 copper atoms. Insertion of these cofactors and assembly of the CCO complex in the inner mitochondrial membrane requires many accessory proteins. Coxl7 is the metallochaperone responsible for delivery of copper to the mitochondria. Once copper has been imported into the mitochondrion, Sco 1 is required for insertion of copper into the CuA site located in Cox2. It has been suggested that Coxl 1 plays a role in the delivery of copper to the CuB site in Coxl. Preliminary evidence indicates that Coxl 1 is a copper-binding protein that is required for the production of active CCO.


The role of supramolecular chemistry in titanium antitumor compounds

Francesco Caruso a, Miriam Rossi b. l s t i tu to di S t ru t tur i s t i ca Chimica, CNR, Box 10, 00016 Montero tondo S taz ione (Rome) ITALY (emai l . f rancesco , caruso@mlib , cnr. it)

b Vassar College, D e p a r t m e n t o f Chemistry, Box 484 Poughkeeps ie NY 12604-484 USA

Common features between the two Ti antitumor compounds that reached clinical trials, Ti(Cp)2Clz (titanocene dichloride) l and Ti(bzac)2(OEt)2 (budotitane). ~ include a fast hydrolysis of their leaving groups (C1 and ethoxide, respectively) and a much slower loss of their complexing ligands (Cp = cyclopentadienyl and bzac = benzoylacetonato, respectively). Recently a tetranuclear Ti-13-diketonato spoecies, 3 related to budotitane, showed selective in vitro antitumor activity, and in vivo increased life span similar to that of budotitane, demonstrating that Ti-polynuclear complexes can be involved in antitumor biological activity. Polynuclear Cp-Ti compounds, obtained frrom hydrolysis, are thus suggested as potential antitumor agents. The insolubility in physiological medium of some of these compounds will be shown to be a problem having viable solutions.

1. Christodoulou C.V., Ferry D.R., Fyfe A.Y. et al., J. Clin. Oncol. 16, 2761-2769 (1998). 2. Schillings T., Keppler B.K., Heim M.E. et al., Invest. New Drugs 13,327-332 (1996). 3. Caruso F., Rossi M., Tanski, J. et al., J. Med. Chem. 43, 3665-3670 (2000)

Acknowledgment: Mary Segue Foundation (MR).

Spin coupling patterns in the MoFe and P clusters of nitrogenase

David A. Case Tim Lovell, Jian Li, Tiquing Liu, and Louis Noodleman Dept. o f Molecu lar Biology, The Scripps Research ].nstitute, La Jolla, CA 92037 USA (e -mai l :case@scr ipps . edu)

The molybdenum-iron protein of nitrogenase contains two types of polynuclear metal-sulfur clusters: the P cluster (with eight irons) and the MoFe cluster (with seven irons and one molybdenum). Based on analyses ofhyperfine interactions, and on extensive density functional calculations, we propose qualitative spin-coupling models for both clusters in several oxidation states. For the MoFe cofactor cluster, in the M N (S=3/2) resting state, we believe that the most likely formal oxidation state corresponds to Mo4+6Fe2+Fe 3+. The spin coupling corresponds to a Mo3Fe cube with S, = 2 antiferromagnetically coupled to a Fe4 cluster with spin 7/2. This is the arrangement favored by DFT calculations, and provides a good account of Fe hyperfine and Mossbauer isomer shift measurements. This spin coupling is analogous in several ways to that found in the P cluster, suggesting some common features in these double-cubanes, even though they have quite different geometries. We have also looked at the character and energetics of states that are one electron more oxidized and one electron more reduced than the resting states, as well as at a variety of states in which both electrons and protons are added to the resting state cluster. These computational results give a picture of likely intermediates on the pathway to formation of both dinitrogen and dihydrogen; a potential pathway for the production of H2 emerges from these calculations. The poster will present both qualitative and quantitative descriptions of the electronic structures of these clusters, and will discuss prospects for future studies of the nitrogenase mechanism.

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