abstracts poster presentations (z11)

Journal of Inorganic Biochemistry 86 (2001) 471

Electron tunneling wires to probe enzyme active sites

Randy Villahermosa", Harry B. Gray ", Jay R. Winkler ", Corinna Hess ~, Michele A. McGuirlL " Department of Chemistry, Beckman Institute, California Institute of Technology, 1200 E.

California Blvd., Pasadena, CA 91125 USA (e-mail. rand@caltech.edu)

A series of electron tumaeling wires have been designed and synthesized to probe deeply-buried enzyme active sites. The tunneling wires are based on donor/acceptor complexes consisting of a [Ru(bpy)3] 2+ (where bpy = 2,2'-dipyridyl) acceptor and 4-methoxy,N.N-dimethylaniline donor. A rigid rod-like phenylene oligomer bridges the donor and acceptor. Mediated by hydrophobic contacts, these electron tunneling wires are able to dock to deeply-buried protein active sites ~hrough substrate chmmels. Utilizing the flash-quench technique, we are able ~o rapidly, < 500 ns, generate a hole at the active site through a radical cation localized on the tunneling wire amine donor. Binding of the tunneling wire through the aniline to the active site of a bacterial amine oxidase, an enzyme that catalyzes the oxidation of amines to aldehydes, has been studied.

// - - ; -"X

1. Klinman, J.P. Chem. Rev. 1996, 96, 2541-2461.

• the National Science Foundation is acknowledged for financial support.

First functional assignment of EIaC

Andreas Vogel, Oliver Schilling, Wolfram Meyer-Klaucke EMB c/o DESY, Notkestr. 85, Building 25A, 22603 Hamburg, Germany (e-mail. vogel@embl-hamburg, de)

A gene annotated as ElaC can be found in E. coli and humans, which shares approximately 50 % homology. Recently, the human EIaC was identified as a prostate cancer susceptibility gene 1. Primary structure analysis of ElaC assumes arylsulfatase activity and a metallo-[3-1actamase fold 2, a mono- or binuclear zinc and iron binding domain. A typical [3- lactamase active site was recently experimentally confirmed by EXAFS data analysis 3. In order to elucidate for the first time the structural and functional features of ElaC, we homologously overexpressed the E. coli ElaC gene. A one step purification procedure yields pure protein of 36 kDa molecular mass. The isolated recombinant protein contains substoichiometric amounts of iron and zinc. Interestingly, ElaC shows no activity towards arylsulfates and glucose-6- sulfate. Moreover, no hydrolytic activity for [3-1actamase and glyoxalase II substrates and para-nitrophenylphosphate could be detected. An enzymatic function of ElaC was detected using a screening procedure utilizing 188 different substrates. Positive reactions were found for the phosphordiesters thimidine-5'-para-nitrophenylphosphate and bis-(para- nitrophenyl)phosphate (bpNPP). Further inspection of the bpNPP activity could strongly correlate the activity with the metal content: the specific activity of the isolated protein of 0.7 + 0.2 U/mg was decreased to 0.13 + 0.03 U/mg after incubation with iron and increased approximately 100 fold after incubation with Zn to 62 i 9 U/mg. Determination of the kinetic parameters of the Zn incubated enzyme resulted in a KM for bpNPP of 3.2 + 0.6 mM and l%~t of 80 ± 6 s -1. For the first time an EIaC protein was isolated and a function assigned. Moreover, the high activation of the phosphodiester activity by Zn suggest this metal to be the physiological cofactor of ElaC.

1. Tavtigian SV, Simard J, Teng DH, et al. Nat. Genet., 27, 172-80 (2001) 2. Melino S, Capo C, Dragani B, Aceto A, and Petruzzelli R., Trends Biochem. Sci.,23,381-2 (1998) 3. Schilling O, Vogel A, and Meyer-Klaucke W, this edition

472 Journal of Inorganic Biochemistry. 86 (2001)

M~issbauer and E P R studies of mononuelear [Fem(N4Py)(qLOOH)] 2+ and IFem(N4Py)(q Z-OO)]+ - models for activated bleomycin

Vladislav Vrfijrna~u ~, Eckard Miinck ~, Raymond Ha b, Lawrence Que Jrb., Gerard Roefles ~, Ben L. Fefinga ~

~Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA. (e-maiL" em40@andrew.emu.edu) bDepartment of Chemsitry and Center for Metals in B ioeatalysis, University of Minnesota, Minneapolis, MN 55455, USA. ~Department of Organic and Molecular Inorganic Chemistry, University of Groningen, Nijenborgh 4, 9747 A G Groningen, The Netherlands

Mononaclear Fe(III)-hydroperoxo species have been proposed as hatermediates in the mechanisms of the antitumor drug bleomycin and a number of mononuclear iron oxygenases and oxidation catalysts. The deprotonation of [Fem(N4Py)(qLOOH)J -'÷ (1) gives [Fel"(N4Py)(q '- OO)]* (2), as unequivocally demonslxated by resonance Raman spectroscopy (v(O-O) - 827 cm" and v(Fe-O) - 495 cm t). We report here M0ssbauer and EPR studies of these Fe(IIi)-hydroperoxo (S=1/2) and Fe(III) peroxo (S=5/2) species. High-spin complex 2 has an isomer shift of 0.61 ram/s, a value similar to those of diiron (llI,III) peroxo complexes. The A-tensor of this high-spin ferric complex was found to be anisotropic; with A~, Ay, Az = -29.5, -29.6, -27.8 MHz. We observed

1 2

similar values for an S = 5/2 Fe(IlI) EDTA peroxo complex. Complex ] is low-spin ferric, with g~, gy, g~,= 2.16, 2.1 I, 1.98, Ax, Ay, A~ = -9.1, +6.1, -52.7 MHz, 6=0.17tarry's, and AEo=l.62 nurds.

The ant i tumor plat inum complexes as an inhibitor of DNA topoisomerase I

Old~ich Vr~na, Viktor Brabec institute, of Btophysics, Academy of Sciences of the Czech Republic, Krdlovopotsk6 t35, CZ-612 65 Brno, CZECH REPUBLIC (e-mail: vrana@ibp.cz)

Type I DNA topoisomerases mediate changes in DNA topology by catalyzing the sequential breakage and rejoining of single phospodiester bond in a DNA strand. These enzymes play essential role during DNA processing including replication, transcription and repair. Topoisomerase I is the cellular target of numerous anficancer drugs, including cainptothecins and some DNA intercalators and minor grove binders. Cisplatinum (eis-DDP) is a widely used anticancer drug which exerts its cytotoxic activity by damaging DNA and some combination chemotherapy regimens have been prompted by a therapeutic synergy between cis-DDP and some topoisomerase inhibitors. The results presented in our contribution demonstrate that some platinum complexes inhibit DNA topoisomerase I not only when added to topoisomerase I before it could interact with its substrate (direct interaction with the enzyme), but also that some Pt- DNA adducts are able to inactivate topoisomerase I when it c/eaves DNA. The results demonstrating the interaction between platinum comp]exes and topoisomerase I inhibitor camptothecin will be also discused.

This research was supported by the Internal Grant Agency of the Academy of Sciences of the CR (grant. no. A7004805/1998 ) and the Internal Grant Agency of the Ministry of Helath of the CR (grant noNL6069-3/2000)

Journal of Inorganic Biochemistry 86 (2001) 473

Monolayers of a de novo designed 4-c~-helix bundlecarboprotein and partial structures on Au(111)-surfaces

H. Wackerbar th , J. Brask , K.J. Jensen, J.U. Nielsen, J. Zhang, J.E.T. Ander sen and J. Uls t rup Department o f Chemistry, Buildings 201 and 207, Technical University o f Denmark, DK-2800 Lyngby Denmark, (e-mail." hw@kemi.dtu.dk)

Mapping of structure and function of proteins adsorbed on solid surfaces is important in a variety of contexts. Spectroscopic, thermodynamic, and voltammetric methods have been central, but electrochemical techniques based on atomically planar single-crystal metal surfaces and in situ scanning probe microscopy techniques such as scanning tunnelling microscopy (STM) have recently been introduced I This has opened new perspectives for structural mapping of immobilized metalloproteins at the single-molecule level. De novo design and synthesis of functional model proteins and peptides have evolved in parallel 2. This strategy promises new test and control of protein folding, and enables construction of isolated protein structural motifs with novel tailored properties. These two strategies towards molecular protein science are combined in the present report.

We present first a synthetic scheme for a new 4-ct-helix bundle carboprotein built on a galactopyranoside derivaUve with a thiol anchor aglycon suitable for surface immobilization on gold. The galactopyranoside with thiol anchor and the thiol anchor alone were prepared for comparison. Voltammetric data for the three molecules adsorbed on single-crystal Au(111)-surfaces show clear reductive desorption features unambiguously caused by adsorption of the molecules via thiolate-Au bonding and monolayer formation. In situ STM of the thiol anchor discloses an ordered overlayer on the Au(111)-surface where domains and single-molecule features can be distinguished. These results hold promise for voltammetric and scanning probe characterization and control of functional natural and synthetic redox proteins towards the single-molecule level.

1. Q. Chi, J. Zhang, J.E.T. Andersen and J. Ulstrup, J., Phys. Chem. B 105, 4669 - 4679 (2001).

2.W.F. DeGrado, C.M. Summa, V. Pavone, F. Nastri and A. Lombardi, Annu. Rev. Biochem., 68,779 - 819 (1999).

The stabilization of la-peroxo copper(II) dimers by hydrogen bonding and steric effects of tripodal ligands

Akira Wada, a Yasu taka Honda, a Shigenori Nagatomo, b Teizo Kitagawa, b Koichiro Jitsukawa, a and Hideki Masuda a,b

a Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan (e-mail.r l 1 ach 02@edsys. center.nitech, ac.jp) b Institute for Molecular Science, Myodaiji, Okazaki 444-8585, Japan

Peroxo copper(II) complexes deserve attention as models for elucidating the structure and function of copper oxidase and oxygenase in biological systems. In order to understand the relationships between electronic and coordination structures of their Cu(II) active sites, the synthesis and characterization of mononuclear azido Cu(II) complexes with novel tripodal ligands having hydrogen bonding NH: group L, tris(6-amino-2-pyridylmethyl)amine (TAPA), bis(6-ammo- 2-pyridylmethyl)(2-pyridyl-methyl)amine (BAPA) and mono(6-amino-2-pyridylmethyl)bis(2-pyridylmethyl)amine (MAP A), have been carried out. Here, on the basis of the spectroscopic and structural studies in the series of [Cu(L)(N3)] +, we examined the hydrogen bonding and steric effects of NH2 substitutents on the stability and electronic properties of trans ~t-l,2 peroxo Cu(lI) dimers [{(L)Cu}:(O2)] 2.. The hydrogen bonding interaction of NH2 groups with the peroxide drastically raised up the thermal stability of [{(L)Cu}2(O2)] 2. These electronic properties, correlated with the n*-to-d CT transitions and peroxide-based stretching vibrations (v(O-O) and v(Cu-O)), depended on the strength of the hydrogen bonds. We concluded that the higher stability of [ {(L)Cu } 2(02)] 2* could be achieved by the strong fixation of the peroxide ion through hydrogen bonding interaction and the formation of the peroxo-copper unit in sterically restricted environment.

474 Journal of Inorganic Biochemistry 86 (2001)

NMR and pulsed ENDOR evidence for thermodynamic stabilization of the dxy electronic ground state in low-spin ferriheme models of heme oxygenase:

implications for the reactivity of the iron(III) hydroperoxide intermediate

F. Ann Walker a, Mario Rivera b, Gregori A. Caignan b, Andrei V. Astashkin", Arnold M. Raitsimring a, Yatjana Kh. Shokhireva b. ~Department of Chemistry, University of Arizona, Tucson, AZ 85721-0041 USA bDepartment of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071 USA

EPR spectra of heme oxygenase (HO), ~ cyt P4502 and ferriheme models bound to an axial HOO- or ROO- ligand 3 are very similar. More important, the sum of the squared g values (Yg 2) in each of these spectra is -14 or less. Since the more common electronic §round state in which the unpaired electron resides in one of the d, orbitals (dx_, or d,~) displays EPR spectra with Yg = 16, this compressed g anisotropy suggested to us that the unpaired electron in the Fem-OOH complex of HO resides in the iron d~ orbital. It is noteworthy that heine models known to possess the

4 I 4 (d~:,d~0 (dxy) electronic structure display large electron and spin density at the porphyrin meso-carbons. 1 Furthermore, ferriheme mod-els with the (d~y) ground state ~yl]ically have very ruffled porphyrin cores, with meso-

carbons alternating -0.6 A above and below the mean plane. Enhanced meso spin density, together with porphyrin ruffling, therefore, might facilitate heme catabolism by HO by positioning two of the meso carbons (either ~x and y, or ~ and 5) closer to the terminal OH of the FemOOH group. A d. w electronic structure also suggests that the regioselectivity of heme oxygenation is dictated by a combination of electronic and steric effects. Pulsed ENDOR and ~3C NMR spectroscopic evidence is presented which show that although the unpaired electron in low-spin ferrihemes containing a ROO- ligand resides in a d~ orbital at 8K, the d~ ground state is strongly favored at ambient temperatures. Variable temperature NMR studies indicate a dynamic system in which a planar d~ porphyrmate ring is in chemical equilibrium with a ruffled ring with a d~y unpaired electron. Because of the similarity in the EPR spectra of the hydroperoxide complexes of HO, cyt P450, HRP and the model complexes reported herein, it is possible that these two ring conformations may also exist in the enzymatic systems. 1 Davydov, R.M.; Yoshida, T.; Ikeda-Saito, M.; Hoffman, B.M.J. Am. Chem. Soc. 1999, 121, 10656. 2 Davydov, R.; Macdonald,I.D.G.; Makris, T.M.; Sligar, S.G.; Hoffman, B.M.J. Am. Chem. Soc. 1999, 121, 10654. 3 Tajima, K.; Jinno, J.; Ishizu, K.; Sakurai,Ohya-Nishiguchi, H. Inorg. Chem. 1989, 28, 709 4 Walker, F.A.; Nasri, H.; Torowska-Tyrk, I.; Mohanrao, K.; Watson, C.T.; Shokhirev, N.V.; Debrunner, P.G.; Scheidt, W.R.J. Am. Chem. Soc. 1996, 118, 12109

The protective role of copper-zinc superoxide dismutase in s. c e r e v i s i a e amino acid metabolism

Mat thew A. Wal lace a, L e e - L o u n g Liou a, Val ter D. Longo b, Joan Se lvers tone Valent ine a, Edith But ler

Gral la a

~' Department of Chemistry and Biochemistry, University of California at Los Angeles(UCLA), 405 Hilgard Ave., Los Angeles, CA 90095-1569, USA (e-mail: matthew@chem,ucla.edu)

b Division of Neurogerontology Andrus Gerontology Center, University of Southern California, 3715 McClintock Ave., Los Angeles, CA 90089-0191, USA

Sacharomyces cerevisiae mutants lacking copper-zinc superoxide dismutase (CuZnSOD, encoded by the SOD 1 gene) exhibit amino acid auxotrophies for lysine, methionine, and, as we now demonstrate, leucine. Furthermore, these auxotrophies can be induced in wild type yeast subjected to the redox cycling drug paraquat, implying that they are due directly to superoxide damage. The lysine and leucine biosynthetic pathways both contain a superoxide sensitive 4 iron 4 sulfur (4Fe-4S) cluster enzyme homologous to aconitase, homoaconitase (LYS4) and isopropyl malate isomerase (dehydratase) (LEU1) respectively. In aerobically grown sodlA yeast cells homoaconitase activity is much lower than in wild type cells, while growth under low oxygen conditions restores homoaconitase activity, even in the presence of cycloheximide. Immunoblots show that native CuZnSOD and its copper chaperone Lys7p are located in the cytosol and the intermembrane space. Homoaconitase is known to reside in the mitochondria so its protection by SODI must take place in the intermembrane space. Isopropyl malate isomerase resides in the cytosol and is likely protected by SOD1 in that location. We conclude that CuZnSOD plays an important role in the protection of 4Fe-4S cluster enzymes such as homoaconitase, in the intermembrane space, and isopropyl malate isomerase, in the cytosol, from oxidative damage.

Journal of Inorganic Biochemistry 86 (2001) 475

Mult inuc lear copper complexes as intermediates of oxidat ive cycl izat ions

Olaf Walter, Michael Ciesielski, Manfred Doering

Institute for Technical Chemistry (ITC-CPV),Forschungszentrum Karlsruhe P.O. Box 3640 76021 Karlsruhe, Germany (e-mail: olaf. walter@itc-cpv.fzk, de)

A growing class of biologically important catalysts are the multicopper oxidases and oxigenases ~, noteworthy members of which include Tyrosinase, Laccase, and ascorbate oxidase. Multinuclear copper enzymes are involved also in the physiologically relevant oxidative heterocyclizations, such as phenoxazinone synthase or sulochrin oxidase. Recently, we could show that methylpyridine substituted Schiff bases undergo copper-mediated oxidative Cyclization forming heterobicycles. 2 This reaction may be carried out with catalytic amounts of copper(II) by air oxygen. Mechanistic studies on this reaction are presented showing that intermediate dinuclear or multinuclear copper complexes should be involved in the oxidation. One isolated intermediate complex (shown in inset) proofs that mixed valent copper(I)/copper(II) species might play a role in the mechanism. Furthermore, we show that these multinuclear copper complexes not only activate the oxygen but influence also the selectivity of the reaction depending on the substitution pattern of the substrates.

1. Solomon, E.I.; Sundaram, U.M.;Machonkin, T.E.; Chem. Rev. 96, 2563, (1996) 2. Doering, M.; Ciesielski, M.; Goerls, H.; J. Inorg. Biochem. 74, 117, (1999)

The Deutsche Forschungsgemeinschaft (SFB 436) is acknowledged for financial support.

Cyste ine-sul fenic acid modif icat ion is essential for the activity of nitrile hydratase

Naoki Watanabe a'b, Hiroshi Nakayama a, Masafumi Odaka a, Yoshiaki Kawano a, Koji Takio a, Nobuo Kamiya a, Teruyuki Nagamune b, Isao Endo a aRIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan (e-mail: ttO6762@mail, ecc. u-tokyo, ac.jp) bDepartment of Chemistry and Biotechnology, School o f Engineering, The University o f Tokyo, Hongo 7-3-I, Bunkyo-ku, Tokyo 113-8656, Japan

Nitrile hydratase (NHase) catalyzes the hydration of various nitriles to their corresponding amides. NHase from Rhodococcus sp. N-771 is an c~[3 heterodimer with a non-heme ferric iron at the catalytic center. The enzyme is known to be stabilized by a competitive inhibitor, n -butyric acid (n-BA) (typically 40 mM), but the role of n-BA has not been identified. Nltase is also known to form a stable inactive complex by the nitrosylation of the iron center, and the nitrosylated enzyme is activated by photo-induced denitrosylation. We have shown the crystal structure of NHase at 1.7 A resolution, and revealed that two out of three cysteine ligands are post-translationally modified (ctCys112-SO2H and c~Cysll4-SOH) ~. Some recombinant NHases having no modification were activated by the oxidation of cxCysll2, showing that these modifications were important for the catalytic activity. In this study, we analyzed the relation between the modifications and the catalytic activity. When NHase was incubated in the absence of n-BA, its activity almost disappeared by about 230 h. Mass spectrometry measurements have shown that c~Cys114-SOH is oxidized to c~Cys114- SOzH as the activity decreases. On the other hand, NHase incubated with 40 mM n-BA showed more than 60 % of the original activity even after 230 h, and c~Cysll4-SOH was oxidized only in about 30 % of NHase. When NHase was incubated under anaerobic condition without n-BA, the activity decreased by about 15 %, and ctCys114-SOH was not oxidized even after 144 h incubation. From these results, we concluded that c~Cys114-SOH is essential for the catalytic activity of NHase, and that n-BA prevents ctCys114-SOH from being oxidized by air.

1. Nagashima S., Nakasako M., Dohmae N., Tsujimura M., Takio K., Odaka M., Yohda M., Kamiya N. and Endo I., Nat. Struct Biol., 5,347-351, (1998)

476 Journal of Inorganic Biochemistry 86 (2001)

Reversible binding of NO to open chain [NzO2 2-] iron(II) chelate complexes. A model for the NO-binding heme protein from the salvia ofRhodniusprolixuJ q

Birgit Weber, Helmar GOrls and Ernst- G, J~tger* Mstitute of inorganic and analytical Chemistry, Friedrick Schiller University dena, August- Bebet Str. 2, 07743, Jena, Germany e-matt: c6webi@gmx.de

iron Porphyrins are involved in various parts of the nitrogen metabolism. As part of a more general program we investigated the reactivity of NO with "bin inspired" iron chelate complexes derived from equatoriai macrocyclic ~N42-1 and open chain [N2022-] chelate ligands. Some iron(II) complexes bind nitric oxide reversibly as shown in figure 1 This behavinur resembles the reversible NO binding of the nitrophorins NP 9 in the salvia of blood-sucking insects. These heine proteins release NO and take up histamine that is released by the victn'n [1]. The reversible binding has been proved by EPR and UV-VIS spectroscopy. Crystals of the ixon(II) complex and the NO-Adduct have been characterized by X-ray analysis.

R COOFa ~ . ~ CGK)Et

P~ Py~i~n

"COOEI COOEr R R

Fi~e I

Ding X. D., Weichsel A., Andersen J. F., Shokhireva T. Kh., Balfour C., Pierik A. J., Averill B. A., Monffort W. R. and Walker F. A , J. Am. Chem. Soc. 121,128-138 (1999)

I would like to thank the Fond der Chemischen Industrie for their financial support.

Copper(II) complexes with [3-ketoenaminic iigands based on aminocarbohydrates and aminoaleohol derivatives of steroids as eatechol oxidase models

Rainer Wegner ~, Manuela Dubs b, Michael Gottschaldt b, Dieter Klemm b, Bruno SchOnecker b and Ernst=G. J~iger ~ ,2 Institute for Inorganic and Analytical Chemistry, Friedrich-Schitler-University Jena, August-

Bebet-Str. 2, D-07743 dena, Germany, (e-mail: c7wera@uni-jena.de) Institute for Organic and Macromolecular Chemistry, Friedrich-Schitter-University dena, tIumboIdtstr. 10, D-07743 Jena, Germany

Copper complexes of tridentate ligands composed of amino alcohol derivatives of natural substances (carbohydrates [1] and steroids P-l, component A) and ~3-ketoenols (component B) are efficient catalysts for the catechol oxidase-iike oxidation of 3,5-di-tert-butylcatechol (dtbc). The influence of different combinations of aminoaleohol and [5- ketoenol component on the reactivity of the resulting copper complexes is discussed.

1 a) Wegner R., Gottschaidt M., G6rls H., J~ger E.-G.~ Klemm

2.

/ - - 'NHz R ~ x j / W

A B

R'-

(,,/

D., Angew. Chem. Int. Ed. Engl., 39, 595 - 599 (2000); b) Wegner R., Gottsehaldt M., GOrls H., J~iger E.-G., Klemm D., Chem.-Eur. J., 7, in press (2001) Wegner R., Dubs M., G6rls H., Robi C., J~iger E.-G., Sch6neeker B., Chem.-Eur. J., 7, submitted

We wish to thank the Deutsche Forsehungsgemeinschafi (SFB 436) for financial support.

Journal of lnorganic Biochemistry 86 (2001) 477

Properties of photogenerated aromatic side-chain radicals in rhenium-modified copper proteins

William A. Wehbi, Angel J. Di Bilio, Brian R. Crane, Jay R. Winkler, Harry B. Gray Beckman Institute, California Institute of Technology, Pasadena, California 91125, U.S.A.

Aromatic amino acid radicals are key intermediates in many biological processes. In our work on electron transfer in proteins, we have developed laser flash/quench methods that potentially could facilitate the study of such highly reactive intermediates in rhenium-modified Pseudomonas aeruginosa azurin (Az). j Phototriggering of Cu 2+ or Zn 2+ Re(Hisl07) azurin generates YI08. , which has been trapped at 77 K and characterized by EPR spectroscopy (see figure). Of special interest is that calculations and experiments on the tlis107 protein show that rate constant for Cu + oxidation via electron transfer through an intervening tyrosine (Cu ~ --) T~, --) Re 2+) is over two orders of magnitude faster than optimized Cu' --) Re 2~ electron tunneling. To test our model, we are investigating the YI08F mutant of Re(CO)3(phen)(His 107)Az.

gz = 2 .0018

332 334 336 338 340 342 H (mT)

1. Di Bilio, A.J., Crane, B.R., Wehbi, W.A., Kiser, C,N., Abu-Omar, M.M., Carlos, R.M., Richards, J.H., Winkler, J.R., Gray, H .B . J . Am. Chem. Soc. 123, 3181-3182 (2001).

The metal binding affinity and solution structure of a novel endonuclease

Joel T. Welch ~ and Sonya J. Franklin b

Department o f Chemistry, University o f lowa, 33 7 Chemistry Building,, 52242, Iowa City, IA U.S.A. (e-mail: joel-welch@uiowa, edu)

b Department o f Chemistry, University o f Iowa, 373 Chemistry Building 52242, Iowa City, IA U.S.A.

By exploiting a near super-imposable turn of two similar yet unrelated protein folds, we have designed a new class of DNA nucleases. The combination of the recognition helices of the engrailed homeodomain with the metal binding loop of calmodulin has generated chimeric peptides that possess both affinity for DNA and hydrolytic metal binding. In the presence of either Ca (II) or Ln 0II), this helix-loop-helix design has a metal dependent structure which shares elements of both parent folds. In solution, this chimeric motif exhibits a complex "back to back" dimerization equilibrium reminiscent of isolated EF-hands. Experiments addressing the thermodynamics, metal binding affinity, and secondary and tertiary structure will be presented in detail. Kinetic analysis of cleavage ofbis-nitrophenyl phosphate, a DNA model substrate, will address nuclease ability.

1. J .T. Welch, M. Sirish, K. M. Lindstrom, and S. J. Franklin, Inorg. Chem., 40 (9), 1982-1984 (2001). 2. Y. Kim, J. T. Welch, K. M. Lindstrom, and S. J. Franklin, J. Biol. Inorg. Chem., 6 (2), 173-181 (2001).

478 Journal of lnorganic Biochemistry 86 (2001)

Nitrido and imido vanadium(V) complexes stabilized by the simple cyclam ligand. Synthesis, structure and reactivity

Thomas Weyhermtiller ", Heike Schucht a, Katsura Mochizuki b, Karl Wieghardt a ~Max-Planck-Institut fiir Strahlenchemie, Stiftstr. 34-36, 45470 Miilheim (Ruhr), Germany

(e-mail.-weyherm@mpi-muelheim. mpg. de bDepartment of Chemistry, Yokohama City University, Yokohama, Japan

Nitrido species of first row transition metal complexes attract much interest as potential nitrogen transfer agents in organic synthesis ~ and as model compounds for high valent metal sites in metalloproteins. Some nitrido complexes of Cr, Mn and Fe have been published but examples of vanadium are still scarce. The complex cis- [LVV(N)(N3)] ÷ 1, quantitatively forms upon illumination of cis-[LVm(N3)2] ÷ under anhydrous conditions (L=l,4,8,11-tetraazacyclotetradecane). Complex 1 is highly reactive and tmmediately forms oxo- and alkoxo compounds in the presence of water and alcohols, respectively. This is in stark contrast to the relatively inert homologues [LMV(N)N3)] n÷, M=Cr,Mn, which do not undergo hydrolytic reactions 2. Reaction of 1 with triflouroacetic anhydride in dichloromethane yields the unusual idmido-amido species cis-[LVV(NCOCF3)(OCOCF3)] ÷ 2. The crystal structure clearly shows that the imido ligand is bound in a linear fashion with a short V-N bond at 1.72 A. One Nitrogen of the macrocyclic ligand is deprotonated and forms a tight amide-V bond.

2 1 . ~

1. J. Du Bois, Craig S. Tomooka, Jason Hong, Erick M. Carreira, Acc. Chem. Res. 1997, 30, 364. 2. Karsten Meyer, Jesper Bendix, Nils Metzler-Nolte, Thomas Weyhermtiller, Karl Wieghardt,

J. Am. Chem. Soc. 1998,120, 7260. b) Karsten Meyer, Jesper Bendix, Eckhardt Bill, Thomas Weyhermfiller, Karl Wieghardt, Inorg. Chem. 1998, 37, 5180.

DNA minor groove binding by multi-nuclear platinum anti-cancer complexes

Nial 3. Wheate and J. Grant Collins School of Chemistry, University College, University of New South Wales, ADFA, ACT 2600

AustraIia (e-mail.n. wheate@adfa, edu.au)

The application of platinum drugs in the treatment of human cancer is mostly reliant on their ability to prevent DNA transcript and replication(I). This is affected by the drugs binding (electrostatically/covalently) to the DNA. Different DNA adducts have different effects. Recently a new type of platinum complex has entered clinical trials; BBR 3464, which is a cationic platinum trimer. In order to understand the increased efficiency of this drug, and others similar to it, it is necessary to understand what type of adducts they form with DNA.

Our group has shown that inert cationic platinum complexes pre- associate in the DNA minor groove and A/T rich regions(2), inducing some conformation changes(2), prevent transcription(3) and show some activity in murine cancers(4). There is still however, debate whether covalent adducts are formed in the minor groove and to date none have

4* H~N [ NH H H N

~N N~FI/ ~ ~.~N ~ ~pt ~ Ci

~' ~NH N MaN I /~ " NH~ P i N J ~ ~ ' ~

2* Cir. oNH H N jCl

H~N H / N - N ~ N H - N NH~

been reported. Recently we have been able to show that one dinuclear platinum complex (d) does form adducts in the DNA minor groove at the N3 of A bases in a variety of oligonucleotides, in some cases the minor groove adducts accounting for 30% of the total adducts formed.

t Reedijk, J. Chem Comm, 7, 801-809 (1996) 2 Wheate, N.J. and Collins, J.G.J. Inorg. Biochem., 78, 313-320 (2000). 3 Wheate, N.J.; Cutts, S.M.; Aldrich-Wright, J.R.; Phillips, D.R. and Collins, J. G. J. Inorg. Biochem.,

84, 119-127 (2001) 4. Wheate, N.J.; Webster, L.K.; Brodie, C.R. and Collins, J.G. Anti-cancer Drug Design, (2001) in press.

Journal of lnorganic Biochemistry 86 (2001) 479

Investigating protein sensitised lanthanide ion luminescence in complexes formed between terbium and the iron binding proteins; human serum transferrin, neisserial ferric iron binding protein and escherichia coli bacterioferritin

Gaye F. White a, Ian Harvey b, Nick E. LeBrun a, Jaqui A. Farrar a, Andrew J. Thomson a, ~School of Chemical Sciences, University of East Anglia, Norwich, NR4 7T J, England bSynchrotron Radiation Source, CLRC Daresburv Laboratory, Warrington, WA4 4AD, England (e-mat1. gaye. wh ite@uea, ac. uk)

This study compares the luminescent properties of complexes formed between terbium (Tb 3.) and the proteins, human serum transferrin (Tti-), recombinant N-lobe transferrin (Tf/2N), Neisserial ferric iron binding protein (Fbp) and Escherichia coli bacterioferritin (Bfr). Tfr, Tf/2N and Fbp perform similar functions as ferric iron transport proteins. The arrangement of amino acid residues that form their metal ion binding sites is very similar. In each case, two tyrosine residues are directly co-ordinated to the metal ion. The complexes they form with Tb 3+ ions bound in place of Fe 3+ ions are being investigated as exemplars of efficient protein sensitised lanthanide ion luminescence. In contrast, the bacterial iron storage protein, Bfr, binds metal ions at sites that do not involve direct contact between the metal ion and aromatic amino acid residues. The luminescent properties of the complex formed between Tb 3+ ions and Bfr are demonstrated and compared to those of Tb2-Tfr, Tb-Tf/2N and Tb-Fbp. We have used X-ray spectroscopy (EXAFS and XANES) to investigate the co-ordination spheres of Tb s+ ions in the Tb-Tf/2N and Tb-Fbp complexes. Analysis of this data is in progress and results of full multiple scattering analysis of the EXAFS together with the XANES will also be presented.

The Engineering and Physical Sciences Research Council, (EPSRC), UK, is acknowledged for fmancial support. We also acknowledge T.A. Mietzner, University of Pittsburgh School of Medicine, and R.T.A. MacGillivray, University of British Columbia, for kindly donating the Fbp and Tf/2N.

O-Atom transfer to thiastearoyl-ACPs by the diiron enzyme stearoyl-acyl carrier protein A 9 desaturase

Robert D. White and Brian G. Fox Department of Biochemistry, University of Wisconsin-Madison, 1710 University A venue, Madison, Wisconsin 53705, USA

The substrate analogs 9-thiastearoyl-ACP and 10-thiastearoyl-ACP were prepared by in vitro enzymatic synthesis and reacted with the reconstituted soluble stearoyl-ACP Z 9 desaturase complex. Electrospray ionization mass spectral analysis of the acyl chains purified from the reaction mixtures revealed that the 10-thia analog was converted to a 10-sulfoxide as the sole product. By use of 180z, the sulfoxide oxygen was shown to derive exclusively from Oz, not from solvent. For reaction of the 9-thia analog, a small amount of 9-sulfoxide (-5% of total products) was obtained with the O- atom again exclusively derived from Oz. These results confirm the ability of the diiron center in the soluble desaturase to catalyze O-atom transfer in the presence of an appropriate substrate analog. For the 9-thia analog only, another molecular species with m/z = 349 amu was identified as the major product of the enzyme reaction (~95%), with the 48 ainu increase in mass consistent with the incorporation of three O-atoms. Isotopic labeling studies using 1802 showed that no O-atom from 02 was retained in (and possibly transferred into) the m/z = 349 product. In contrast, a partial incorporation of labeled O-atoms was obtained when the enzyme reaction was performed in -75% 18OH2. Identification of the m/z = 349 ainu species by synthesis of authentic standards is in progress. Reaction mechanisms accounting for sulfoxide products of the stearoyl-ACP Z 9 desaturase reaction are discussed.

This work was supported by National Institutes of Health Grant GM-50853 to B.G.F.R.D.W. was a trainee of the NIH Institutional Biotechnology Pre-Doctoral Training Grant T32 GM08349.

480 Journal of Inorganic Biochemistry 86 (2001)

Antiproliferative activity of the Co(lI), Ni(II), Cu(II), Pd(II) and Pt(II) complexes of 2-(4-thiazolyl)benzimidazole(thiabendazole)

Marek Wi~niewski a, Adam Opolski b, Joanna Wietrzyk b ~Institute of Chemistry Pedagogical University, Chqcihska 5, 25-020, Poland bInstitute of Immuno logy and Exper imenta l Therapy, Polish Academy of Sciences, Weigla 12, 53-

114 Wroctaw, Poland

Complexes of 2-(4-thiazolyl)benzirnidazole(thiabendazole, THBD) with Co(II), Ni(II), Cu(II), Pd(II) and Pt(II) of general formula MLz(NO3)2H20 obtained and characterized by elemental analyses, IR and far IR spectroscopy, magnetic examination and conductonometry. X-ray crystal structure of the copper(I1) complexes has been determined. The in vitro cell proliferation inhibitory active of these compounds was examined against human cancer cell lines A549 (lung carcinoma), HCV-29T (urinary bladder carcinoma), MCF-7 (breast cancer), T47D (breast cancer), MES-SA (uterine carcinoma) and HI-60 (promyelocytic leukemia). Pt- THBD has been found to exhibit an antileukemic activity against line H1-60 cells matching that of an arbitary criterion.

The geometry of Cr(VI) species in the system [3d metal ion-2,2'bpy-CrO4 v]

aAgnieszka Wojciechowska, b Wieslawa Bronowska, CAdam Pietraszko, d Zbigniew Staszak, a

Maria Cieslak-Golonka,

" Department of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 50-37 Wroclaw, POLAND (e-mail. wojciechowska@ichn.ch.pwr, wroc.pl)

b Department of Physics, Wroclaw University of Technology, POLAND c Institute of Low Temperature and Structure Research, Polish Academy of Science, Okolna 2,

59-950 Wroclaw, POLAND dFaculty of Computer Science and Management, Wroclaw University of Technology, POLAND

27,

The coordination model explains a decrease (even of 90 %) of the genotoxicity of metal ion complexes containing the CrO42 entity bonded to the central metal ion [ 1-2]. This work is an attempt to fmd the hierarchy of the factors responsible for the chromate geometry in the complexes. We have studied the title systems in various experimental conditions, like M:L:Cr(VI) ratio, counter anions, acidity and have observed structurally and spectroscopically the positions of the chromate ions. The variety of the chemical compositions isolated and the diversity of the chromate coordination will be discussed in details.

1. Szyba K., Cieslak-Golonka M., Gasiorowski K., BioMetals, 5, 157 (1992) 2. Gasiorowski K., Szyba K., Wozniak D., Cieslak-Golonka M., Rzepka-Matemy M., BioMetals, 11, 175-181 (1998)

The authors would like to thank the Wroclaw University of Technology (No. 342117) and Polish Committee for Scientific Research (Grant No. 3T09A 09218) for financial support of this work.