abstracts poster presentations (y)

Journal of Inorganic Biochemistry 86 (2001) 361

Crystal structure of the complex of photoreactive nitrile hydratase with substrate analogue, cyclohexyl isocyanide

M a s a k i Noj i r i a, Y o s h i a k i K a w a n o a, M a s a f u m i O d a k a b, I sao E n d o b, and N o b u o K a m i y a a

~'Division of Bio-Crystallography Technology, R1KEN Harima institute~Spring-& 1-1-1, Kouto, Mikazuki-cho, Sayo-gun, Hyogo, 679-5148, JAPAN (e-mail. [email protected])

bBiochemical Systems Laboratory, RIKEN(The institute of Physical and Chemical Research), 2-1, Hirosawa, Wako-shi, Saitama, 351-0198, JAPAN

Nitrile Hydratase (NHase), capable of catalyzing hydration of nitrile compounds, is well-known as one of the most industrially successful enzymes. It is currently used for industrial production of acrylamide more than 30,000 tons / year in the world. The enzyme consists ofc~ and [3 subunits and contains a mononuclear low-spin non-heine iron or non- corrinoid cobalt at the active site. We have revealed the crystal structure of Fe-type NHase at 1.7 A resolution (1). Very interestingly, the sulfur atoms of c~Cys 112 and etCys 114, ligands of the non-heme iron, are post-translationally modified to cysteine-sulflnic (c~Cys 112-SO2H) and cysteine-sulfenic acid (~xCys 114-SOH), respectively (1). However, the role of the post-translational modifications has not been revealed.

Recently, we have succeeded in determining the crystal structure of the complex of NHase with a substrate analogue, cyclohexyl isocyanide. The analogue directly bound to the non-heme iron atom at the active center of NHase, and the hydroxyl group of c~Cys 114-SOH was disappeared. Although it is unclear whether the group was really removed from or disordered around the sulfur atom of c~Cys114, it is strongly suggested that the hydroxyl group of ctCys114-SOH is an important group in the reaction mechanism. Additionally, new two broad bands were appeared at around 650 and 850 nm in the vlsible spectrum of the complex. The spectrum was similar to that of complex of NHase with a substrate, isovaleronitrile. This result indicates that the structure of complex with cyclohexyl isocyanide is similar to that of complex with isovaleronitrile. 1. Nagashima S., et al. Nat. Stmct. Biol., 5, 347-351, 1998

Preliminary X-ray crystallographic studies on two oxidation states of recombinant P. stutzeri cytochrome c4

Allan Norgaard a, Pernille Harris b, Sine Larsen b, Hans E.M. Christensen a. " Department of Chemistry, Technical University of Denmark, Kemitorvet, 2800 Kgs. Lyngby,

Denmark (e-mail: [email protected]) b Centre for Crystallographic Studies, Copenhagen University, Universitetsparken 5, 2100

Copenhagen O, Denmark

Cytochrome c4 is a bacterial di-heme electron transfer protein] As revealed by a previously determined 2.2 A 2 resolution structure, the protein from P. stutzeri consists of two similarly folded domains each containing a heme group.

Due to its relative simplicity, it serves as a model system for studying cooperative features in electron transfer proteins. With two heme groups, the basis for cooperative behaviour is present, and at the same time the number of physical parameters forming part of a detailed characterization are within reach of a complete disentanglement.

Recombinant P. stutzeri cytochrome c 4 has been cocrystallized with cryoprotectants. The oxidation states of the protein crystals have been verified spectrophotometrically. Low-temperature X-ray data sets have been collected at I7-11, MAX-lab in Lund. The data extend to 1.25 A (oxidized form) and 1.35 A (reduced form), and should enable analysis of the structural changes accompanying electron transfer and an estimation of the reorganization Gibbs free energy of the protein.

1. Andersen N.H., Christensen H.E.M., Iversen G., Norgaard A., Schamagl C., Thuesen M.H. and Ulstrup J., "Cytochrome c4" in "Handbook of Metalloproteins", Eds. Messerschmidt A., Huber R., Poulos T. and Wieghardt K., John Wiley & Sons Ltd, 2001.

2. Kadziola A. and Larsen S., Structure, 5,203-216 (1997).

The Technical University of Denmark is acknowledged for a Ph.D. scholarship to A.N.

362 Journal of Inorganic Biochemistry 86 (2001)

Post-translational modification of photoreactive nitrile hydratase in the photoactivated state

Masafumi Odaka, Yoshiaki Kawano, Hiroshi Nakayama, Masanari Tsujimura, Koji Takio, Nobuo Kamiya, and Isao Endo RIKEN(The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan (e-mail: modaka@postman, riken.go jp)

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to the amides. The enzyme is used for the industrial production of acrylamide, but the reaction mechanism remains unknown. NHase from Rhodococcus sp. N-771 shows a unique photoreactivity: The enzyme is inactivated by nitrosylation of the catalytic iron center, and activated by photo- driven NO release. Recently, the crystal structures in photoactivated ~ (2.6 A resolution) and nitrosylated z (1.7 A resolution) states were reported. Both structures are very similar and the ligands atoms are identical, showing that large conformational change does not occur during photoactivation. However, there were important differences between the structures around the iron center. In the former, no modification was observed, whereas c~Cys112 and c~Cys114 were post-translationally modified to Cys-SOzH and Cys-SOH in the latter. To understand this problem, we determined the crystal structure of NHase in the photoactivated state as follows. (A): The photoactivated NHase was crystallized in the presence of a competitive inhibitor, 40 mM n-butyric acid. In this crystal, both ctCys112 and ctCys114 existed as Cys- SO2H, and the activity decreased into less than 1% of the native NHase. (B): The nitrosylated NHase was crystallized and then activated by light irradiation at 100K or room temperature. In both temperatures, the sixth ligand, NO, was replaced by a water molecule, showing that the enzyme was photoactivated in the crystals. Crystal structure analyses and mass spectrometry measurements showed that Cys114 exist as Cys-SOH in the crystal irradiated at 100 K, but Cys114 was partially oxidized to Cys-SO2 H when the crystal was irradiated at room temperature.

~. Huang W et al., Structure, 5,691-699 (1997) 2. Nagashima S. et al., Nat. Struct Biol., 5,347-351, (1998)

Copper(II) - tetrahydropterin interactions. Complex formation and oxygen activation in aqueous solution

Akira Odani a, Kiichiro Kimura b, Yasuhiro Funahashi c, Osamu Yamauchi d Research Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, JAPAN (e-mail:[email protected])

b Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, JAPAN C Department of Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, JAPAN 'lUnit of Chemistry, Facul~' of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, JAPAN

Tetrahydropterins are known as cofactors for various metalloenzymes such as aromatic amino acid hydroxylases, which introduce an oxygen atom into the aromatic ring of the amino acids by activating dioxygen. However, little is known about the mode of metal-tetrahydropterin interaction at the active site and the 02 activation mechanism. It remains unclear why the reaction center of tyrosine hydroxylase has a water molecule coordinated with the dthydropterin cofactor uncoordinated in the solid state structure. To e~tp.,.I shed light on the metal-tetrahydropterin interaction we studied the complex formation and redox reaction in the ternary Cu(II)-L-6,7-dimethyl-5,6,7,8-tetrahydropterin(H4DMP) Hq ?-L3 systems (L = EDTA, 1,10-phenanthroline(phen), etc.) by pH titrimetry. Under anaerobic O-t--CU-N" -k conditions HD4MP formed a temary complex with Cu(II)-L below pH 8, and at higher pH a N~/- ~I~'H %'-ff redox reaction occurred to form the Cu-O(4) bond. In contrast to this Cu(phen)(HD4MP) HzN~NJ"N,J ~

H formed without redox reactions even under aerobic conditions. 02 absorption was observed H4pterin.._O 2

for solutions of H4DMP and Cu(phen)(H4DMP). These results suggested formation of a quaternary complex, Cu(II)-phen-HaDMP-O2, where H4DMP-O2 may be a 4a-OOH form of pterin. Without Cu(II)- phen, 02 absorption was very slow, and HaDMP-O2 decomposed to give H202, which indicates that Cu(II)-phen may play an important role in 02 activation and stabilization of the 02 adduct. Cu(II)-phen-H4DMP-O2 hydroxylated di-tert- butyl-phenol to form di-tert-butyl-catechol, supporting that the activated form of tetrahydropterin is the 4a-OOH form stabilized by metal coordination.


Magnetic circular dichroism of symmetry and spin forbidden transitions of high- spin metal ions. The use of ab initio methods in calculation of MCD spectra

Vasily S. Oganesyan and Andrew J. Thomson Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of

East Anglia, Norwich, NR4 7TJ, UK.

We previously presented a general method of analyzing magnetic circular dichroism (MCD) spectra and magnetization curves of high-spin metal ions for spin-allowed transitions ~. We have now extended this approach to cover the cases of spin- and symmetry-forbidden transitions 2. Extraction of information content about the electronic structure of the ground state can be obtained through the analysis and correlation of the positions, signs and intensities of the MCD bands and magnetization curves of these transitions. This together with the analysis of the bands from allowed transitions provide complimentary insight into metalloprotein active sites. The casting of the theory in terms of the irreducible tensor method allows full advantage to be taken of any symmetry elements and simplifies multi-electron calculations. The theory is valid over the entire range of magnetic field strength and therefore allows the information content of spectra over the full field and temperature range to be exploited. The method is applied to the analysis of the recorded MCD spectra and magnetization curves of the lowest energy spin-forbidden ligand field transitions of pseudo-tetrahedral high-spin Fe(III), S-:5/2, in the protein rubredoxin from Methanobacterium thermoautotrophicum (strain Marburg9. The predicted signs, intensities and magnetization curves for these transitions are in excellent agreement with experimental data. We also show that when the anisotropy of the ground state is larger than the Zeeman splitting the MCD of both spin-forbidden and allowed transitions can become comparable in magnitude. Hence caution is needed in order to avoid the misinterpretation of experxmental results. The use of first principles calculation methods such as CASSCF/PT2 and post-SCF C13 for prediction and analysis of MCD and absorption spectra of metalloproteins is also demonstrated. I V.S. Oganesyan, S.J. George, M.R. Cheesman and A.J. Thomson. J. Chem. Phys., 110, 762, 1999. 2 V~S. Oganesyan and A.J. Thomson, J. Chem. Phys., 113, 5003, 2000. 3. B.D. Roos, K. Andersson and M.P. Fulscher, Chem. Phys. Lett. 192, 5,1992. We thank BBSRC for support of the Center for Metalloprotein Spectroscopy and Biolog

Does the catalytic cycle of cytochrome P450 involve a second oxidant: a theoretical investigation of the hydroperoxo hypothesis.

Frango l s Ogl i a ro , S a m P. de Visser, S a s o n Sha ik

Department of Organic Chemistry and the Liser Meinter-Minerva Center for Computational Quantum Chemistry The Hebrew University of Jerusalem, 91904 Jerusalem Israel.

The microsomal P450s are the major enzymes involved in oxidations of physiological importance. The general consensus, holding at least for hydroxylation and epoxidation, is that an high-valent oxoiron(IV) porphyrin radical cation is most likely the sole reactive species capable of monooxygenation. Iq Very recently, experimental work on P450 and model systems by independent groups have challenged this view. [2'3'41 It is suggested that, depending of the experimental conditions and the nature of the substrate, the hydroperoxo complex formed during the catalytic cycle of P450 is also able of inserting an oxygen atom in alkane and alkene. To address this question, we studied using B3LYP/LACVP calculations the model compound [FePorSHOOH] -t. We characterize it with respect to a variety of complexes including its neutral form ([FePorSHOOH]), the primary oxidant Compound I ([FePorSHO]), as well as a the axial ligand-free [FePorOOH] q (q = 0, -1) systems. In addition, we discuss its capability to epoxide ethane either by direct oxygen addition or by an OH+ insertion.

1. Ortiz de Montellano P.R., Ed. Cytochrome P450: Structure and Mechanism and Biochemistry, 2 "ded,; Plenum Press: New York (1995).

2. Newcomb M., Shen R., Choi S.-Y., Toy P.H., Hollenberg P.F., Vaz A.D.N., Coon M.J.J. Am. Chem. Soc. 122, 2676-2686 (2000).

3. Nam W., Lira M.H., Moon S.K., Kirn C. J. Am. Chem. Soc. 122, 10805-10809 (2000). 4. Collmann J.P., Chien A.S., Eberspacher T.A., Brauman, J.I.J. Am. Chem. Soc. 122, 11098-11100 (2000).

The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.


The synthesis of S-phosphorodithiolate analogs of di- and oligonucleotides with sulfur in 3'- or 5'-bridging position

Andrzej Okruszek, Magdalena Olesiak, Wojciech J.Stec Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 L6d~, Poland, (e-mail:okruszek@bio. cbmm.lodz.pl)

The S-phosphorothiolate analogs of oligonucleotides, in which 3'- or 5'-bridging oxygen atom in the phosphodiester linkage is replaced by a sulfur atom, are indispensable tools for investigating the biochemistry of nucleic acids, and in particular in studying the involvement of metal ions in phosphoester transfer reactions. L2 In order to synthesize corresponding phosphorodithioate analogs with one of the sulfur atoms in either 3 '-3.4- or 5'- bridging position, two independent approaches were employed. The 5'-S-phosphorodithiolate analogs were prepared by alkylation of nucleoside 3'-O-phosphorodithioates 5 with 5'-bromo(or iodo)-5'-deoxynucleosides. The appropriately protected dithymidyl analog, after transformation into 3'-O-phosphoramidite derivative, was successfully employed for introduction of 5'-S-phosphorodithiolate dinucleotide segment into the preselected position of oligonucleotide. The 3'-S-phosphorodithiolate analogs were prepared from appropriately protected 3'-mercapto-3'-deoxynucleosides, after their transformation into 3'-S-(2-thiono-l,3,2-oxathiaphospholane) derivatives. These were reacted with 5'-hydroxy- nucleosides in the presence of DBU to give the product of oxathiaphospholane ring cleavage and the formation of 3'-$- phosphorodithiolate linkage. The reaction performed with diastereomerically pure oxathiaphospholane substratc confirmed full stereospecificity of ring opening condensation.

1. Piccirilli J.A., Vyle J.S., Caruthers M.H. and Cech T.R., Nature, 361, 85-88 (1993) 2. Weinstein L.B., Jones B.C., Cosstick R. and Cech T.R., Nature, 388, 805-808 (1997) 3. Cosstick R. and Vyle J.S., Nucl. Acids Res., 18, 829-835 (1990) 4. Yoshida A., Sun S. and Piccirilli J.A., Nature: Struct. Biol., 6, 318-321 (1999) 5. Okruszek A., Olesiak M., Krajewska D. and Stec W.J., J. Org. Chem., 62, 2269-2272 (1997)

Towards PSII analogs driven by ruthenium photophysics

Jerry A. Olsson ~, Catherine E. HousecrofP, Ebbe Nordlander b, Edwin C. Constable ~School of Chemistry, University of Birmingham, United Kingdom (e-mail: jj0911 @bham. ac. uk) blnorganic Chemistryl,Chemical Center Lund University, Sweden)

A number of models complexes have been prepared in an attempt to develop models for photosystem II (PSII) in green plants. As replacement for the chlorofyll photosensitizer, we have used Ru 11 Iris- bipyridyl- or bis-terpyridyl complexes linked to a "flee" bipyridyl or terpyridyl moieties via spacers of varying lengths. Manganese(II) has been covalently linked to the "free" bipyridyl/terpyridyl moieties. The use of different ruthenium centres and spacers have made it possible to make assumptions about the way and how easily manganese is coordinated through self-assembly to "free" bipyridyl or terpyridyl. It was found that the length of the spacer is crucial due to deactivation of the "free" end by ruthenium. The use of different ruthenium centres has also shown to be important; the ruthenium bipyridyl centre deactivates the "free" ligand less than the terpyridyl analogue.


Biological activity studies of complex compounds with nitronyl nitroxides

O. O p r e a b, A, Z a r k a d i s a I. D r a g u t a n c, T. N e g r e a n u - P i r j o l d

a Univers i ty o f Ioannina, P.O. Box 1186, 45110 IOANNINA, GREECE h Univers i ty "POLITEHNICA "" Buchares t , Gh. Pol izu 1, BUCHAREST, ROMANIA, (e-mail: Ovidiu 73@yahoo. corn) c "Costin D. Nen i t e scu " I n s t i t u t e o f Organic Chemistry , S p l a i u l I n d e p e n d e t e i 202 B, 71141 BUCHAREST, ROMANIA dFaculty o f Med ic ine and Pharmacy , OVIDIUS Universi ty , Ioan Voda 58, CONSFANTA, ROMANIA

The complex compounds (Co IJ, Ni n, CuII, La IH, Ce m, Gd llz) action toward some bacteria cultures was tested both in liquid and solid medium. The substances were solved in liquid medium or laid on the agar-agar solid medium surface pre- inseminated with tested bacteria. When the testing substance has antibacterial action, in the higher concentration area than M. I. C. (minimum inhibitory concentration) the inseminated bacteria are inhibited. By measuring inhibition diameter the "'bactericide power" of the tested substances is established the "bactericide power" of the tested substances.

We chose as method the testing on solid culture medium made by impregnation of several Millipore filter paper microdiscs. The biological activity of these compounds was studied toward Corynaebacterium diphteriae, Streptococci hemolyticus group A, Streptococci faecalis, Bacillus cereus, Pseudomonas aeruginosa set. I/1, Pseudomonas aeruginosa ser. IX, Escherichia coli, Salmonella group B, Shigella flexneri, Yersinia enterocolitica, Candida albicans.

This work is supported by the Romanian Ministry of Education and Greek Ministry of Research

New complex compounds with free stable radicals

O. Oprea b, A. Zarkadis a, I. Dragutan c a Univers i ty o f l o a n n i n a , P.O. Box 1186, 45110 IOANNINA, GREECE b Univers i ty " 'POLITEHNICA '" Bucharest , Gh. Pol izu 1, BUCHAREST, ROMANIA, Ovidiu 73@yahoo. corn c "Costin D. N e n i t e s c u " I n s t i t u t e o f Organic Chemistry , S p l a i u l l n d e p e n d e t e i 2 0 2 B, 71141 BUCHAREST, RO MAN IA

The design of molecular magnets is one of the challenges in the 21st tasks for chemists in this area of research is the synthesis and study of suitable new bridging ligand systems that provide efficient electron exchange interactions between spin carriers. Nitronyl nitroxides (NITs) are known as building blocks for the preparation of magnetic materials. In most NIT-metal complexes the metal is coordinated only by the N-O moiety and electron-withdrawing coligands. In order to obtain compounds of higher dimensionality, we have synthesized further functionalized NITs and were able to isolate a series of new transition metal complexes (Co 1I, Ni tJ, Cu H) with these ligands. The electronic properties of the compounds are discussed.

century coordination chemistry. One of the main

Acknowledgment: this work is supported by the Romanian Ministry of Education and Greek Ministry of Research


Synthesis and characterization of 4f complexes with nitronyl nitroxides

O. Oprea b, A. Zarkadis a, I. Dragutan c University o f loannina, P.O. Box 1186, 45110 IOANNINA, GREECE

~' University " P O L I T E H N I C A " Bucharest , Gh. Polizu 1, BUCHAREST, ROMANIA, Ovidiu [email protected] c "Costin D. N e n i t e s c u " I n s t i t u t e o f Organic Chemistry, S p l a i u l l n d e p e n d e t e i 202 B, 71141 BUCHAREST, ROMANIA

In the metal-radical approach nitronyl nitroxide organic radicals has been successfully used as (bis)monodentate bridging ligands to build low dimensional systems exhibiting magnetic ordering at low temperatures. We have synthesized complex compounds of La m, Ce m and Gd nl with 2-ethyl-4,4,5,5-tetramethyl-4,5-dihydro-lH- imidazolyl-l-oxy-3-oxide and 2-(1 •-methy•pyridine-•-)-4•4•5•5-tetramethy•-4•5-dihydr•-•H-imidaz••y•-•-•xy-3-•xide. One of the appealing features of molecular magnets is that they may have properties which are not usually met in classic magnets. The electronic and magnetic properties of the resulting complexes will be discussed.

Acknowledgment: this work is supported by the Romanian Ministry of Education and Greek Ministry of Research

D4-symmetric calixporphyrins

S_jgrid Ostermann, Johann Schlrgl, Bernhard Kr/iutler* Inst i tute o f Organic Chemistry, University o f lnnsbruck, Innrain 52a, A-6020 Innsbruck, Austria

We report here a synthetic route to D4-symmetric Zn- and Co(II)- calixporphyrins. The relevant stereochemistry was introduced in a highly diastereoselective Diels-Alder-reaction of 2,6-di-tert- butylanthracene with fumaric acid di-(-)menthyl ester, catalyzed by aluminium chloride. 1 The porphyrin framework was built up as described, z Applying Horeau's principle, the chiral calixporphyrin was calculated to be present in an enantiomeric excess of at least 2.5 x 10~S:l. The NMR-spectral properties of the two calixporphyrins were compared and characteristic differences in chemical shifts due to the presence/absence of a paramagnetic Co(II)-center were found. Catixporphyrins may act as rigid hosts for organic guest molecules 2 and chiral biconcave porphyrins are useful as chiral shift reagents 3.

1.

2 .

3.

Schwenninger R., Ramondenc Y., Wurst K., Schlrgl J., Kr~iutler B., Chem. Eur. J., 6, 1214 - 1223 (2000) Schl6gl J., Krgutler B., Synlett, S1,969 - 971 (1999)

\

M = Z n M = CoOl)

Schwenninger R., Schl6gl J., Maynollo J., Gruber K., Ochsenbein P., Btirgi H.-B., Konrat R., Kniutler B., Chem. Eur. J., in press

This work was supported by the Austrian National Bank (project P-7889).


Transition metal complexes of peptides containing chelating imidazole-N donors

Kata l in 0 s z a, Ka ta l in V f i m a g y a , I m r e S6vf ig6 a, H e l g a S ~ l i - V a r g h a b, G i o v a n n i M i c e r a c, Dan ie l e S a n n a d

'~Department of Inorganic and Analytical Chemistry. University of Debrecen, H-4010 Debrecen, P.O.Box 21, Hungary(e-mail. [email protected])

hResearch Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest 112, P.O.Box 32, Hungary

CDepartment of Chemistry, University of Sassari, Via Vienna 2,1-07100 Sassari, Italy Jlnstituto C.N.R. per l'Applicazione delle Tecniche Chimiche Avanzate ai Problemi Agrobiologici.

Via Vienna 2, 1-07100 Sassari, Italy

The ligands containing two or more imidazole rings linked via aliphatic carbon chains can potentially mimic the binding sites and catalytic activities of histidine containing metalloenzymes. Moreover, specific enzyme inhibitors may be obtained by attaching the bis(imidazolyl) residues to peptides having the preferred amino acid sequence for the peptide cleavage. ~-3 The coordination chemistry of a series of peptide molecules containing the bis(imidazol-2-yl)methane chelating agent at the C-termini revealed that the donor functions of the peptide backbone can compete with chelation of the bis(imidazolyl) residue at pH 7. The presence of the terminal amino group promotes the successive deprotonation of amide nitrogens, while imidazole residues act as additional donor sites or bridging ligands. A histamine-like or GlyHis- like coordination can be formed with the histidyl residues depending on the position of the histidine amino acid. The presence of the imidazolyl side chains facilitates the deprotonation of pyrrole-N(1)H donor functions at higher pH.

i. K. Vfimagy, I. S6vfig6, W. Goll, H. Stili-Vargha, G. Micera and D. Sanna, Inorg. Chim. Acta, 283,233-242 (1998) 2. K. Varnagy, I. S6vfig6, H. Sfili-Vargha, D. Sanna and G. Micera, J. Inorg. Biochem., 81, 35-41 (2000) 3. K. 0sz, K. Vfimagy, I. S6vfig6, L. Lennert, H. Stili-Vargha, D. Sanna and G. Micera, New J.Chem., 25,700-706 (2001) The Hungarian Scientific Research Fund (OTKA T29141) and Jfinos Bolyai Research Grant (No. BO/00099/98) are acknowledged for financial support.

Equilibria between rotamers of [Pt(tmen){d(GpG)}] + studied by means of molecular modeling, NMR and circular dichroism

Diana E. Over, Miguel A. Elizondo-Riojas, Gildas Bertho, V6ronique Bas, Jiri Kozelka Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR CNRS 8601, 45, rue des Saints-POres, 752 70 Paris Cedex06, France. e-mail.'over@biomedicale, univ-paris5 fr

The structural changes induced upon coordination of cisplatin to the dinucleotides GpG or dGpG have been widely studied by NMR and CD techniques. However, the dynamic character of these adducts due to the interconversion between different head-to-head (HH) and head-to-tail (HT) conformations via rotation about the Pt-N7 bond makes the interpretation of spectra difficult, since these processes are rapid on the NMR time scale. In order to slow down this rotation, sterically hindered chelating diamine ligands were employed by several groups including ours. ~ We have specifically studied the reaction of dGpG and GpG with asymmetrically hindered platinum complexes containing substituted ethylenediamine derivatives. In the case of the complex [Pt(dmen)(GpG)]" (dmen = N,N=dimethyl- ethylenediamine) the guanine cis to the dimethyl group cannot freely rotate about the Pt-N7 bond, while the other base is free to turn. In fact, two HH isomers are formed, one of which could be resolved into two slowly interconverting rotamers. Upon addition of another methyl group to the ligand to form N,N,N' trimethylethylenediamine (tmen), the base cis to the NHMe group is now constrained to one single orientation, minimizing the steric repulsion with the methyl group and at the same time allowing for a NH-O6 hydrogen bond. Therefore, each HH isomer of [Pt(tmen){d(GpG)}] + can only be in equilibrium with one head-to-tail conformer and the equilibrium should be sufficiently slow so that each form may be resolved by NMR. Surprisingly only two major products are formed in the reaction of [PtC12(tmen)] with dGpG which were identified as two different HH isomers, one of which has been shown by NMR to be in equilibrium with a small quantity of the corresponding HT conformer. The two isomers were separated by HPLC, completely characterized by NMR, and the CD spectra were recorded. Stereochemical reasoning combined with molecular modeling were used to explain the relative stabilities of the various conformers formed in this reaction.

(a) Williams, K.M.,,Scarcia, T., Natile, G., Marzilli, L.G., Inorg.Chem., 40, 445-454, (2001); (b)Elizondo-Riojas, M. A. Bas, V., Kozelka, J., JBIC, 5, 45-50, (2000).


Syntheses and characterization of metal(III) complexes with NzS3-type ligands containing amido nitrogens

Tomohiro Ozawa, Takayuki Goto, Koichiro Jitsukawa, Hideki Masuda Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, 466-

8555, Nagoya, JAPAN(e-mail: [email protected])

Nitrile hydratase (NHase) is a metalloenzyme that hydrolyzes a nitrile compound to the corresponding amide. The structures of the NHases with Fe(III) ion has been recently determined.t The Fe(III) ion is surrounded with two amido nitrogens of a peptide main chain and three sulfur atoms, in which two sulfurs of the /--.--SH three were posttranslationally modified to sulfmyl and sulfenyl groups. Some enzymes ( with Co(III) instead of Fe(III) are known and its coordinating environment is expected ) <-

to be the same as the Fe(II I )one because of the homology of their sequences. Thus, ~ " H bS~ the investigation on formations and characterizations of the metal(III) (especially Fe(III) and Co(III)) complexes with NzS3-type ligands containing amido nitrogens are SH H interested as the model studies of the NHases. L1

In this study, compounds, L1 and L2, were synthesized as the active site model of NHase. Ligand L1 contains two amido nitrogens and three thiolates as the coordination sites, while for L2 one of the three thiolate groups of L1 is replaced by a thioether group. Spectroscopic and structural characterization of their metal(III) (Fe(III) or Co(III)) complexes will be discussed. ; sH L2 HS 1.Huang W., Jia J., Cummings J., Nelson M., Schneider G., and Lindqvist Y., Structure, 5,691 (1997). 2.Nagashima S., Nakasako M., Domae M., et. al., Nat. Struct. Biol., 5, 347 (1998).

Synthesis and reactivity study of trinuclear Cu(II)-complex of 2,2"-selenobis(2,4-di-t-butylphenol) ligand

T.K.Paine, T.Weyhermtiller, E.Bill, K.Wieghardt and P.Chaudhuri Max-Planck-Institut fiir Strahlenchemie, Stiftstrafle 34-36,D-45470 Miilheim an der Ruhr,

Germany, (e-mail." [email protected])

In the course of investigating the reactivity of potential radical stabilizing metal complexes, we report here a trinuclear Cu(II) complex of tridentate bisphenol ligand. The structurally and spectroscopically characterized trinuclear Cu(II)- complex 1 (tert. butyl groups are not shown) behaves differently in different solvent systems and undergoes structural changes. In Dichloromethane-Acetonitrile solvent mixture it forms biphenol along with a dinuclear Cu(II) complex of modified ligand . This change occurs through radical pathway. ( ~ - o k ~ / ~ Catalytic Reactivity: /~ , . . . -o RCH2R' ~- RCH(OH)R' + RC(O)R' / / ~/s~--Cu Cu~e\ )=X

The accompanying structural changes, radical formation and /(~-O\/cu .~ ~J °"ff'~° x%k-~ reactivity studies on substrates are discussed. CH,CN'[~.(~"~

~4


Bis triazine copper complexes: the relationship between structure, EPR spectral parameters and superoxide radical scavenging activities

C.G. P a l i v a n a a n d B .A . G o o d m a n b

'~ University of Bucharest, Faculty of Physics, Dept. of Atomic Physics, Bucuresti-Magurele, P. O. Box. MG- 1 1, ROMANIA, e-mail:cpalivan@ olimp.fiz, infim, ro

" Scottish Crop Research Institute, lnvergowrie, Dundee, DD2 5DA, UK

Superoxide dismutase (SOD) and related copper complexes have been proposed as pharmaceutical agents to control levels of the superoxide free radical anion ( 0 2 ) during pathological processes. The high molecular weight of the natural proteins, however, limits their membrane permeability, and for this reason related low molecular weight copper complexes are being considered for use as anti-inflammatory and anti-viral drugs. Copper(II) complexes with triazine ligands have been shown to possess SOD activity, but there are considerable variations within groups of closely related complexes ~. The coordination sphere around the copper ion in some families of symmetric and asymmetric bis triazine complexes with SOD activity has been investigated using a combination of EPR spectrometry and molecular dynamics calculations ~. The complexes all have tetragonal symmetry, but with various degrees of distortion. In addition, they may be either four-coordinated with two nitrogen and two oxygen (2N20) atoms bound to the copper, or six-coordinated with a 4N20 arrangement as a result of N-coordinated ligands. The results suggest that a structure involving 6-membered chelate rings with a 2N20 square planar coordination favours SOD activity. Considerably lower activity was observed in planar complexes with 5- or 7-membered chelate rings, and in complexes with 6-membered chelate rings having distortions from the square planar arrangement caused by bulky ligands. Low activity was also observed in complexes with tridentate ligands

l. B.A. Goodman, C.G. Palivan, C. Cristescu, Polyhedron, 1995, 14(17-18), 2523-2535; C.G. Palivan, H.M.N. Palivan, B.A. Goodman, C. Cristescu, Appl. Magnetic. Resonance, 1998, 15(3-4), 477-488.

The formation of copper complexes with hypericin, in solutions: An EPR Study

C.G. Palivan a, G. G c s c h c i d t a a n d L. Weincr b University of Basel, Institute for Physical Chemistry, Klingelbergstrasse 80, CH-4056 Switzerland, [email protected]

~JDepartment. of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.

In order to improve the use of hypericine in photodynamic therapy, limited by its restricted absorption wavelengths (540- 610nm), both the synthesis of derivatives and the formation of hypericin-metal complexes, who could induce a red shift in the absorption spectra, were considered 1,2 In the present study we are trying to characterise in more detail the way of complex formation in various fluid media, by using EPR spectrometry. This can provide information about the geometry of the first sphere co-ordination sphere around the metal as well as the nature of the ligand ions involved in. it. Even more, EPR parameters together with the absorption wavelengths are considered in a ligand field approach,:asst~mmg a D4h syrnlTletry around the metal, in order to obtain the covalence coefficients that characterise the unpaired electron delocalisation in the first sphere co-ordination region. The proposed binding region for the metal was the carbonyl-hydroxyl region of hypericin where repel of the proton could induce a red shift and a change of the co-ordination around the metal, depending on the solvent, was put into evidence.

1. S. Rahimipour, C.G.Palivan, D. Freeman, F. Barbosa, M. Fridkin, L. Weiner, Y. Mazur, G. Gescheidt, Photochem. Photobiol, 2001, in press

2. M. Naris, P. Jardon, J. China. Phys., 1994, 91, 99-112.


The Activation of 02 by the Trinuclear Cu Cluster in Multicopper Oxidases

A m y E. Pa lmer a, L i l iana Quin tanar a, Scott Severance b, Tzu-P in W a n g b, Danie l K o s m a n b, Edward I.

So lomon ~

"Department of Chemistry, Stanford University, Stanford, CA, USA, 94040 b Department of Biochemistry, State University o f New York, Buffalo, NY, USA, 14214

Multicopper oxidases utilize a type 1 Cu and a type 2/type 3 trinuclear Cu cluster to couple substrate oxidation to the reduction of 02. In the overall mechanism, the type 1 Cu accepts electrons from substrate and transfers them to the trinuclear cluster where 02 binds and is reduced to 2~=~_ _, H20. Studies on the activation of O, by multicopper oxidases have been facilitated by replacement of the T1 Cu in laccase with spectroscopically silent and redox inactive ~., .~i\o ~f¢~°" Hg 2+ (T1Hg-Lc). Reaction of reduced T1Hg-Lc with O2 produces a peroxide level \ .x intermediate which bridges the T2 and T3 coppers] Spectroscopic (EPR, CD, MCD) k.2)(~-~ __ ! ~- and kinetic (solvent isotope effect, 16/1802 isotope effect) studies reveal that decay of this / ' intermediate involves reductive cleavage of the O-O bond. 2 This process is 2~c~m~ exceptionally slow because of the large Franck Condon barrier associated with a one o-o reactlon coordinate (rather than two) electron transfer. These studies have also been extended to T1 depleted (T1D) Fet3 and a mutant (H126Q) in which the trinuclear Cu cluster is perturbed. The nature and kinetic behavior of the peroxide intermediate in T 1D and H 126Q are compared to gain further insight into the activation of 02 by the trinuclear Cu cluster.

i. ShinW.,etal. ,J. Am. Chem. Soc., 118,3202-3215(1996) 2. Palmer AE, Lee SK, Solomon EI, J. Am. Chem. Soc. (in press)

Acknowledgment: (N1H DK 31450)

Flavocytochrome c3 from Shewanellafrigidimarina: investigating the proton pathway by site directed mutagenesis

K.L. Pankhurs t a, R . M o y s e y a, C.S. Miles b, E.L. Ro the ry a, M.K. Doherty a, G.A. Reid b, S.K. C h a p m a n a

Department of Chemistry, University o f Edinburgh, West Mains Road, Edinburgh, EH9 3J J, U.K b lnstitute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh,

EH9 3JR, U.K.

Flavocytochrome c3 is the fumarate reductase from the marine bacterium Shewanella frigidimarina ~. It catalyses the reduction of fumarate to succinate in the terminal step of the anaerobic respiration pathway. This process involves the transfer of 2 electrons and 2 protons. Arg 402 has been shown to be the active site acid 2. Protons are supplied to the active site by Arg 381 and Glu 378. This proposed proton pathway has been investigated by site directed mutagenesis.

Solvent isotope studies on the mutants indicated that Arg 402, Glu 378 and Arg 381 are involved in proton transfer. Kinetically these residues have been shown to be vitally important for enzyme function.

FAD

Fumara

Glu 378 ~ ; A r g 402

rg 381 "-~'~

1.Taylor, P.; Pealing, S.L.; Reid, G.A.; Chapman, S.K.; Walkinshaw, M.D., Nature Structural Biology, 6, 1108-1112, (1999). 2 Doherty, M.K., Pealing, S.L., Myles, C.S., Moysey, R., Taylor, P., Walkinshaw, M.D., Reid, G.A., Chapman, S.K (2000) Biochemistry, 39, 10695-10701.

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