abstracts poster presentations (m)

Journal of Inorganic Biochemistry 86 (2001) 241

Mg(II) and Ca(II) complexes of 5- and 6-uracilmethylphosphonate (5Umpa 2- and 6Umpa 2-) in aqueous solution

R o l f Gr iesse r a, Cr i s t6ba l F. M o r e n o - L u q u e a, Jus tyn Ochock i b, H e l m u t Sigel a

"Insti tute of Inorganic Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland (e-mail." [email protected])

b Institute of Chemistry, Medical University, PL-90-151 L6d~, Poland

Derivatives of 5- and 6-uracilmethylphosphonate, m combination with Cisplatin, prolong the ~ ) x survival time of mice with lymphoid leukemia) Since metal ions may be involved in the H 3

biological effect of these phosphonate derivatives, we determined the stability constants of the [ complexes of Mg 2+ and Ca 2+ (= M 2+) with 5Umpa 2- and 6Umpa 2- in aqueous solution (pot. pH y titrations; 25 °C; I = 0,1 M, NaNO3). 2 Based on log KMM(R.PO3) v e r s u s pg~H(R_PO3) straight-line H plots (R-PO~ = simple phosphate monoester or phosphonate ligands with a non-interacting 5Ompa2-: X=--CH2P~- y .-. --.H residue R), it is shown that M(5Umpa) and M(6Umpa) have the stability expected on the basis of 6Ompa2-: x-----H the basicity of the phosphonate group in 5Umpa 2- and 6Umpa2-; i.e., these ligands may be Y=--CH2PO~3- considered as simple analogues of nucleotides. In the higher pH range deprotonation of the uracil residue occurs with formation of M(5Umpa-H)- and M(6Umpa-H)- complexes. Based on comparisons of various acidity constants it is shown that M(5Umpa) is especially acidic, i.e., the M(5Umpa-H)- species are especially stable. This increased stability is attributed to the formation of a 7-membered chelate involving the phosphonate group and the carbonyl oxygen at C4 (after deprotonation of the (N3)H site). The formation degree of this chelated isomer reaches about 45% for Mg(5Umpa- H) and Ca(5Umpa-H)-. There was no indication for chelate formation in the M(6Umpa-H) species.

1. Ochocki J. and Graczyk J., Pharmazie, 53,884-885 (1998) 2. Moreno-Luque C.F., Gricsser R., Ochocki J. and Sigel H., Z. Anorg. Allg. Chemie, in press (2002)

Supported by the Swiss Nat. Sci. Found., the Swiss Fed. Off. for Educ. & Sci. (COST D20), and the Med. Univ. of L6d~.

An investigation of the mechanism of Ru(III) abstraction of NO from hydroxamic acids:

Darren M. Griffi th, Cel ine J. Marm i on , Ter ry M u r p h y and Kev in B. Nolan.

Department o f Chemistry, Royal College of Surgeons in Ireland, 123, St Stephen's Green, Dublin 2, Ireland.

Hydroxamic acids are important in biology and medicine. They are used as constituents of antibiotics, growth factors, tumour inhibitors and cell-division factors. They also act as siderophores for iron(III), potent and selective itflaibitors of enzymes such as ureases, and matrix metalloproteinases, and as hypotensive reagents, i

It was recently reported for the first time that Ru(III) readily abstracts NO from hydroxamic acids. Furthermore it has been shown that hydroxamic acids cause vascular relaxation in rat aorta by activation of the iron-containing guanylate cyclase enzyme. 5 However the mechanism of NO abstraction from hydoxamic acids is still unknown.

Using ruthenium(HI) as an NO scavenger we report herein an investigation into the mechanism of NO abstraction from hydroxamic acids by a ruthenium(III) complex, K[Ru(Hedta)C1].2H20. In doing so we hope to play a pivotal role in establishing their mode of action within biological systems.

1. B. Kurzak, H. Kozlowski and E. Farkas, Coord. Chem. Rev.,1992, 114, 169. 2. C.J. Marmion, T. Murphy, J. R. Docherty and K. B. Nolan, Chem Commun., 2000, 1153-1154.

242 Journal of Inorganic Biochemistry 86 (2001)

Structural studies in solution of nitrogenase complexes based on nucleotide analogues and heterologous cross-reactions

J. Gt inter G r o s s m a n n a, S. S a m a r H a s n a i n a, F a r i d o o n K. Y o u s a f z a i b, R o b e r t R. E a d y b

"Synchrotron Radiation Department, Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, UK (e-mail. j [email protected])

b Department of Biological Chemistry, John Innes Centre, Colney, Norwich, NR4 7UH, UK

Transient electron transfer complexes formed between the two component proteins of nitrogenase are an essential intermediate species during catalysis. Stable but inactive transition-state complexes of nitrogenase components are formed during turnover in the presence of AlE 4" or BeFx. Stable, but active complexes with novel substrate reduction patterns are formed between some combinations of MoFe-protein and Fe protein isolated from different organisms. The determination of low resolution structures using synchrotron X-ray scattering reveals changes in macromolecular conformation of proteins in different complexes in solution. Since the molecular conformation of the component 1 proteins from nitrogenase depends on the metal-cofactor content we were able to isolate a 1:1 complex of which a low resolution structure at -20A resolution was obtained, j It is clear that only one Fe-protein is bound and that this takes place at the c~-subunit of the MoFe protein where the metal cofactor is present. The changes observed in the conformation of the Fe-protein in this 1 : 1 complex appear to be similar to those previously reported for the 1:2 complex where both cofactors are occupied and the Fe-protein is bound to both subunits. 2 Moreover, our scattering studies of the stable but active complexes revealed the conformation of the heterologous and first active nitrogenase complex between Cp2 and Kpl. In addition the Cp2-Avl complex has been isolated and investigated in solution. Uniquely, gel filtration experiments showed the complexes to be of 1:1 ratio, indicating the two Cp2 binding sites of MoFe protein have different affinities.

I. Grossmann J.G., Hasnain S.S., Yousafzai F.K. and Eady R.R., J. Biol. Chem.. 276, 6582-6590 (2001) 2. Grossmann J.G., Hasnain S.S., Yousafzai F.K., Smith B.E., Eady R.R., Schindelin H., Kisker C., Howard J.B.,

Tsuruta H., Muller J. and Rees D.C., Acta Cryst., D55,727-728 (1999)

On the way to the investigation of chiral organometallic compounds with bioactive substituents in biological systems

M a r c A. Grund l a, A. S t e p h e n K. H a s h m i

a Insti tutf i ir Organische Chemie, Johann Wolfgang Goethe-Universitiit Frankfurt a.M, Marie- Curie-Str. 11, 60439 Frankfurt a. M., GERMANY ([email protected])

In the past years, organometallic compounds received considerable attention in pharmaceutical studies, e.g. as diagnostic tools, l As a result there is a growing interest in studying their behaviour in o n nO biological systems, o ~ ~

Pallada-trans-tricyclo[4.1.O.O2'4]heptanes (PTHs) 1_ display properties which meet the basic requirements for application under biological conditions, e.g. they are air-stable and water soluble. 2 Moreover, as a formally unsaturated and chiral compound, interesting interactions with biomolecules can be expected, o ~ "P d "~----o

In order to investigate the influence of bioactive subsfituents e.g. aminoacids or \ f - - nucleobases on the chemical and biological properties of PTHs, we were looking for a way o R ! RO to introduce these groups into the PTH structure.

Here we will present acid and enzyme catalyzed transesterification as an efficient tool for altering the PTH structure. The reaction enables us to introduce linker groups into the PTH that allow coupling to various bioactive compounds.

1. N. Metzler-Nolte, Angew. Chem. Int. Ed. Engl., 40(6), 1040-1043 (2001) 2 W.H. Soine, C. E. Guyer and F. F. Knapp, Jr., J. Med. Chem.., 27(6), 803-806 (1984) 3. A .S .K . Hashmi, F. Naumann, R. Probst and J. W. Bats, Angew.Chem. Int. Ed. Eng., 36, 104 - 106 (1997)


Coordination changes of the heme group in mutated hemophore HasA from Serrat ia m a r c e s c e n s : analysis by EPR spectroscopy and redox potentiometry

B r u n o Guig l ia re l l i a, A n d r 6 F o u m e l a, Marce l A s s o a, Pa t r ick Be r t r and a, P ier re B i a n c o a, CIar isse D e n i a u b, Mur i e l D e l e p i e r r e b, A n n e L e c r o i s e y b

"BioOnergOtique et Ing~nier ie des Protdines, CNRS et UniversitO de Provence , 31 Chemin Joseph Aiguier, 13402 Marse i l le , F R A N C E

bLaboratoire de R~sonanee MagnOtique NuclOaire, Ins t i tu t Pasteur, 28 rue du Doc teur Roux, 75724 Paris, F R A N C E

Hemophores are small extra-cellular proteins produced by some Gram-negative bacteria to provide them with iron. These heme acquisition systems (HasA) extract the heme group from hemoglobin and release it to the outer membrane receptor HasR. The tridimensional structure of the HasA protein from S. marcescens was recently determined (1) and revealed an unusual His(32)-Tyr(75) axial coordination of the heme group, with a second His(83) residue hydrogen bonded to the hydroxyl of Tyr(75). In order to understand the molecular mechanism enabling the binding and the release of the heme group by HasA, these residues were modified by site-directed mutagenesis. The EPR study of the mutants showed that the heme group which is low spin in the wild type protein, changes to high spin in H32A, and exhibits a statistical mixing of high spin and low spin states in H83A and Y75A. The zero field splitting parameters of the high spin states were deduced from the temperature dependence of the EPR signal, and an unusual thermally induced spin transition was observed around 30K in the Y75A mutant. Redox properties of the heine group in the mutants were determined by cyclic voltammetry and redox titrations monitored by UV-visible spectroscopy. The influence of the strenght of the hydrogen bond involving the axial oxygen ligand on the electronic properties of the heme group are discussed in the light of these results.

1. Arnoux, P., Haser, R., Izadi, N., Lecroisey, A., Delepierre, M., Wandersman, C., Czjzek, M., Nat. Struct. Biol., 6, 516- 520 (1999)

Evaluation of the [4Fe-4S] 1+/° reduction potential of the nitrogenase Fe protein

Maolin G u o a, Filip Sulc b, C h a d Iml 'noos b, Patrick J. Farmer b, Barbara K. Burgess a ,l Department of Molecular Biology and Biochemistry, b Department of Chemistry, University of California, lrvine, CA

92697, USA, (e-mail. [email protected])

Azotobacter vinelandii nitrogenase consists of two metalloproteins: the Fe protein and the MoFe protein. The Fe protein is a dimer of two identical subunits bridged by a single [4Fe-4S] cluster which transfers electrons to the MoFe protein, which can subsequently reduce the substrate. The [4Fe-4S] cluster can exist in three oxidation states: 2+, 1+ and 0 in vitro. ~ It has long been known that the [4Fe-4S] 2+~+ couple has a reduction potential of ca. -300 mV (versus SHE). In 1994 Watt and Reddy 2 reported that the Fe protein could be reduced to the 0, all-ferrous state using methyl viologen with a reduction potential o f - 460 mV vs. SHE. It was stated that the +1/0 transition did not result in a change in the UV/Vis spectrum and to date no spectroscopic characterization of that protein has appeared, 2 We were unable to reduce the Fe protein below the +1 state using methyl viologen, however, we have produced a "pink" all-ferrous form of the Fe protein using a variety of reductants. This "pink" all-ferrous protein has since been characterized by M6ssbauer, UV/Vis, CD, EPR (S=4), MCD and x-ray absorption spectroscopies and by x-ray crystallography.l'3 Here we have studied the redox properties of the [4Fe-4S] +/° couple of the Fe protein by examining its reactions with various reducing agents with a wide range of reduction potentials, as well as by using mediated electrochemistry. Interactions of various reduced methyl viologen derivatives (with E0' - 440 to - 780 mV vs. SHE) with the [4Fe-4S] ÷ Fe protein did not yield the "pink" [4Fe- 4S] ° state. However, Cr(II)-EDTA (E0' ca. -1000 mV vs. SHE), Ti(III)-citrate and Ti(III)-EDTA (E0' ca. -800 mV vs. SHE) which readily reduce these methyl viologen derivatives, can also reduce the [4Fe-4S] + Fe protein to the [4Fe-4S] ° state showing that the E °' is far more negative than - 460 inV. Results from electrochemical titrations will be presented. 1. Angove H.C., Yoo S.J., Mtinck E. and Burgess B.K., J. Biol. Chem., 273, 26330-26337 (1998) 2. Watt G.D. and Reddy K.R.N., J. Inorg. Biochem., 53,281-294 (1994) 3. Strop P., Takahara P.M., Chiu H.J., Angove H.C., Burgess B.K., Rees D.C., Biochemistry, 40, 651-656 (2001) The National Institute of Health is acknowledged for financial support. M.G. thanks the ICBIC10 for awarding a grant for young SBIC members and the University of California-Irvine for a Faculty Career Development Award.


P450 system from Acinetobacter calcoaceticus EB104: Induction, preparation, characterization and interaction of components

N. Gupta, O. Asperger Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Leipzig, Talstrasse 33, D 04103 Leipzig, Germany (e-mail: nishll5@hotmail, com)

University of

The P450-dependent monooxygenase system 1 from Acinetobacter calcoaceticus EB104 will be used as a model system for the cell free reconstitution of such enzymes for o-hydroxylation of n-alkanes. Here we report the purification of P450 along with its parmer electron transfer components ferredoxin (Fd) and ferredoxin reductase (FdR) on a preparative scale. Optimized cultivation procedures in 10-1 fermentor batches with nutrient broth as carbon source and n- hexane as inducer resulted in biomass containing 0.4-nmol P450/mg dry weight. P450, Fd and FdR were purified using repeated DEAE anion-exchange and gel filtration chromatography from a single batch of cell free extracts. Properties (absorbance and fluorescence spectra, extinction coefficient, prosthetic group etc.) of Fd and FdR were determined.

With respect to interaction studies we have found that Fd and FdR form a complex with stoichiometric molar ratio of 1:1. NADH-2, 6-dichlorophenol reductase activity of FdR is stimulated by addition of Fd, which further increases with NaCI up to 0.020 M, followed by a decrease at higher salt concentration. NADH-dependent cytochrome c reduction also exhibits same trend in NaC1 dependence of the activity with a maximum at about 0.020 M NaC1 in 50-raM Tris-C1 buffer, pH7.5. Fd-dependent cytochrome c reduction activity of FdR decreases by about 40 %, when reductase is treated at 45 ° for 15 minutes. The presence of Fd largely protected FdR against inactivation. When Fd:FdR ratio is varied by elevating Fd concentration with FdR held constant, an increasing rate of cytochrome c reduction is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via a protein-protein complex of Fd and FdR. Apart from these experiment we have indications of stable stoichiometric complex formation between homogenous Fd and partially purified FdR by gel filtration methods. Further studies to investigate interaction of Fd and FdR together and with P450 are in progress.

1. Asperger, O.; Mueller, R. and Kleber, H-P. (1985) Developments in Biochemistry, Vol.27, 447-450.

Financial support by German Research Council (DFG) in the frame of "Postgraduate Training Programme: Mechanisms and Application of Non-Conventional Oxidation Reactions" is gratefully acknowledged.

Biochemical and structural studies of the tungsten containing glyceraldehyde-3- phosphate oxidoreductase from Pyrococcusfuriosus

P.-L. H a g e d o o m a, J .H. Slits a, J .M. C h a m o c k b, C .D. G a r n e r c, W . R . H a g e n a

aKluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands (e-mail: p.l. hagedoorn@tnw, tudelft, nl) ~CLRC Daresbury Laboratory, Daresbury, Warrington Cheshire, WA4 4AD, UK CDepartment of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK

Glyceraldehyde-3-phosphate oxidoreductase (GAPOR) is involved in the glycolytic pathway of the hyperthermophilic archaeon Pyrococcus furiosus I. The enzyme contains a tungsten-(bis)pterin cofactor and a [4Fe-4S] cluster as prosthetic groups. GAPOR has been previously characterized by EPR spectroscopy and electrochemistry 2. The redox potentials of the W 6+/5 , W 5+/4+ and [4Fe-4S] 2+/+ couples have been determined and suggested an electron transfer pathway from the substrate via the tungsten center to the [4Fe-4S] cluster. To obtain more insight into the mechanism of GAPOR, the kinetic properties of the enzyme and the structure of the tungsten center were studied in more detail.

Steady-state kinetic studies have revealed that the thermal degradation products of glyceraldehyde-3-phosphate (GAP), mainly methylglyoxal, do not interfere with the activity of GAPOR. Furthermore GAPOR activity has been found to be strongly pH dependent with a maximum at pH 9. A kinetic isotope effect kH/k o of 1.66 has been found.

W L(III)-edge EXAFS studies of GAPOR at different redox potentials reveal that the tungsten(VI) is coordinated by two oxo-ligands and that structural changes in the first coordination sphere of the tungsten center occur upon reduction.

1. Mukund S., Adams M.W.W., J. Biol. Chem., 270, 8389-8392 (1995) 2. Hagedoom P.L., Freije J.R., Hagen W.R., FEBS Lea., 462, 66-70 (1999)


Reactions and reduction of platinum(IV) anticancer complexes

M a t t h e w D. H a l l a, M u r r a y S. D a v i e s b, C a r o l y n D i l l o n a, S u e B e m e r s - P r i c e b, T r e v o r W. H a m b l e y a

~Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, N.S.W. 2006, AUSTRALIA (e-mail." [email protected], edu.au)

bDepartment of Chemistry, The University of Western Australia, 35 Stirling Hwy, Crawley, Perth, W.A. 6009, AUSTRALIA

A number of platinum(IV) complexes have entered clinical trials, but none has entered widespread clinical use. The antitumour activity of platinum(IV) complexes is dependent upon a number of important steps. The structure-activity relationships and mechanism of action of platinum(II) drugs have been investigated extensively, I and while the aquation and speciation of platinum(II) anticancer drugs has been recognised as a crucial factor in activity, corresponding studies of platinum(IV) drugs have not been undertaken previously. We have followed the aquation of cis-[PtCla(15NH3)2] by [~H,~SN] 2D HSQC NMR to investigate the kinetics of aquation of this complex, and to identify the aquated species. The cytotoxic target of platinum anticancer drugs is generally agreed to be DNA. However, it has not previously been verified experimentally whether these drugs localise in the cell nucleus. Determining whether reduction of platinum(IV) drugs occurs primarily extracellularly or intercellularly is also a focus of our research. XAFS analysis of platinum(II) and platinum(IV) complexes has also been undertaken to ensure the complexes are stable under bright synchrotron sources. This work is preparative to analysing whole cells and cell sections by SRIXE to compare the total drug concentrations of cisplatin and three platinum(IV) complexes in cells, and identify areas of localistion within cells.

1. Hambley T.W., Coord. Chem. Rev., 166, 181-223 (1997)

The authors thank the Australian Synchrotron Research Program, Australian Research Council and the University of Sydney Cancer Research Fund for financial support.

Theoretical studies on the reaction mechanisms of the nickel-iron and iron only hydrogenases

Michael B. Hall, Zexing Cao, Hua-Jun Fan, Shuhua Li, Shuqiang Niu, and Lisa Thomson Department of Chemistry, Texas A&M University, College Station, Texas 77843 USA

The catalytic cycles for H 2 oxidation in [NiFe] and [Fe-only] hydrogenases have been investigated through density

functional theory (DFT) for a wide variety of redox and protonated structures of the active site models, (CO)(CN)2Fe(~t-

SMe)2Ni(SMe)2 and L(CO)(CN)Fe((~-SCH2)2X)(~t-CO)Fe(CO)(CN)(SMe), respectively. For the [NiFe] modeling, DFT calculations on related complexes are used to build a calibration curve for the

calculated CO bond distances and the measured IR stretching frequencies. By combining this calibration curve with the DFT calculations on the active site model, the redox states and structures of active site have been determined: Ni-B is a Ni(III)-Fe(II) species, Ni-SI(a) is a Ni(II)-Fe(II) species, Ni-SI(b) is protonated on a terminal sulfur, Ni-R is a Ni(II)- Fe(II) with H 2 bound at Fe, and Ni-C is a Ni(III)-Fe(II) species with a F e - - H - - N i bridge. The latter species returns to Ni-SI through a Ni(I)-Fe(II) intermediate. Dihydrogen activation on the Fe(II)-Ni(III) species is more favorable than on the corresponding Ni(II) or Ni(I) species. Our final proposed structures are consistent with IR, EPR, and ENDOR measurements and the correlation coefficient between the measured CO frequency in the enzyme and the CO distance calculated for the model species is 0.905. Recent results on models for Ni-A, and Ni-SU suggest a (~t-OH) and a (~t- OH2) species, respectively. The unconstrained geometry for the H2-cleavage transition state shows a remarkable structural resemblance to the active site in the enzyme crystal structure.

For the [Fe-only] modeling, full frequency calculations on models for the active site and for well-characterized complexes show that observed and catalytically active redox species in the enzyme must correspond to Fe(II)-Fe(II), Fe(II)-Fe(I), and Fe(I)-Fe(I). Furthermore, when X is NH rather than CH2, a single Fe and this N create a very favorable thermodynamic path for the heterolytic cleavage of H2. The H2-cleaved species shows an unusually short "dihydrogen

bond", F e - - H . . . . H - -N .

246 ,, Journal of lnorganic Biochemistry 86 (2001)

The theoretical transition state structure of a model complex bears a striking resemblance to the active site structure of DMSO reductase

Michael B. Hall and Charles Edwin Webster Department of Chemistry, Texas A&M University, College Station, TX 77843-3255 USA (e-mail." [email protected])

Molybdenum containing enzymes are ubiquitous in nature and fulfill a variety of biological catalytic functions. Dimethylsulphoxide (DMSO) reductase catalyzes the oxygen atom transfer from the substrate DMSO. Recently, Lim, Sung, and Holm have shown that a relatively simple inorganic complex [Mo[S2Cz(CH3)z]2OR] l (R=C6Hs) can perform similar chemistry, even though its ligands are severely truncated compared to those of the enzyme. Here, we report density functional calculations on the structures and energies of a model system (R=CH3). Remarkably, the arrangement of the sulfur ligands calculated for the freely optimized transition state, which is quite different from that calculated for the model 's reactant or product, closely resembles the arrangement reported for the crystal structures of the enzyme. The geometric constraint provided by the enzyme not only lowers the reaction barriers but significantly reduces the exothermicity of the S to Mo oxo transfer.

The crystal stucture of nitrous oxide reductase (NzOR) from Paracoccus denitrificans at 1.6 ~ resolution

Tuomas Haltia a, Kieron Brown b, Mariella Tegoni b, Christian Cambillau b, Matti Saraste c, Kristina Djinovic-Carugo d. aInstitute of Biomedicine~Biochemistry, University of Helsinki, P. O. Box 63, FIN-O0014

Helsinki, Finland (e-mail. [email protected]) bArchitecture et Fonction des Macromolecules Biologiques, CNRS, 13402 Marseille, France CStructural and Computational Biology Programme, EMBL, Heidelberg, Germany aStructural Biology Laboratory, Sincrotrone Trieste in Area Science Park, 34012 Basovizza, Italy

Each monomer of dimeric N2OR comprises 2 domains: a C-terminal cupredoxin domain carrying the electron entry site CUA, and an N-terminal J3-propeller domain which hosts the active site Cuz. The electrons are transferred from CUA to CUz across the subunit interface. Cuz is a tetranuclear Cu cluster in which the Cu ions are ligated by seven imidazoles. Cu's I, II and III have two histidine ligands each, whereas CulV is ligated by a single imidazole and an oxygen. Cuz contains S 2- bridging the four Cu's. The absorption spectrum of a blue form of NzOR shows bands at 480, 540 and 640 nm, and around 800 nm. Another form that separates from blue N2OR during purification has a pinkish-purple colour and lacks the absorption band at 640 nm. The loss of this absorption band can be caused by treatment of blue N2OR with H202 or CN. Reduction with ascorbate abolishes the bands at 480, 540 and 800 nm that are attributed to Cua, but does not influence the 640 nm band. Consequently, the 640 nm band appears to arise from Cuz which is partially reduced in blue N2OR. EPR spectroscopy shows that 2 Cu's/blue N2OR monomer are EPR visible. The EPR spectrum of the CN-- treated blue form has an intensity of about 2 spins/monomer. The structure of N2OR soaked with C N suggests that 1 Cu from Cuz has been lost. Addition of H202 to blue NzOR results in a type 2 Cu EPR (total spin conc. of 2-3 spins/monomer). Reduction with ascorbate yields a broad EPR with a spin conc. of ca. l/monomer. This spectrum arises from a Cu 2+ within Cuz.


Estrogen derived steroidal metal complexes; agents for cel lular delivery of metal centres to estrogen receptor-posit ive cells

• b * Mike Harmon a*, Alexander Jackson, a Julie Davis, b Richard J. Plther, Alison Rodger a* Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK (email: m.j. hannon@warwick, ac. uk)

b Nycomed Amersham plc, Amersham Laboratories, Little Chalfont, Bucks, HP7 9LL, UK

Targeted cellular delivery of drugs to specific tissues requires harnessing and applying of molecular-level recognition events prevalent in (or specific to) the desired tissue type. Tissues rich in estrogen receptors (ERs), which include many types of breast cancer, accumulate molecules that have high binding affinities for these receptors. Therefore, molecules that (i)

Me OH

M = Pd, Pt, Re, Zn, Ni

bind to the ER; (ii) have favourable cellular transport properties and (iii) contain a second functionality (such as a centre that may be used for diagnostic imaging or medical therapy) are exciting synthetic targets in the field of drug delivery. We have prepared a range of metallo-estrogens based on 17c~-ethynylestradiol and examined their binding to the ER both as isolated receptor, and in whole cell assays (ER positive MCF-7 cells). All the compounds prepared and tested exhibit effective binding to the estrogen receptor and are delivered across the cell membrane into MCF-7 cells. In the whole cell assays, despite their monocationic nature, the palladium and platinum complexes prepared exhibit similar (and even enhanced) receptor binding afffmities compared to their corresponding neutral free ligands. It is unprecedented for a higher ER binding aff'mity to be observed for a cationic complex than for its metal-free ligand. 1. Jackson A., Davis J., Pither R.J., Rodger A. and Hannon M.J., Inorg. Chem., in press (2001)


Dimethylsulf ide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the D M S O reductase family of

molybdenum enzymes

Graeme R. Hanson b Christopher A. McDevitt a, Alastair G. McEwan a aDepartment of Microbiology & Parasitology, School of Molecular and Microbial Sciences and bCentreforMagnetic Resonance, The University of Queensland, St Lucia 4072, Australia. (e-mail: Graeme.Hanson@cmr. uq.edu.au)

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the N- terminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB).

Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.

XSophe, a computer simulation software suite for the analysis of electron paramagnet ic resonance spectra.

Graeme R. Hanson a, Christopher J. Noble a, Kevin E. Gates b, Kevin Burrage b ~Centre for Magnetic Resonance and bDepartment of Mathematics and The University of Queensland, St. Lucia, Queensland, Australia, 4072. Email: Graeme. [email protected]

The XSophe computer simulation software suite consisting of a daemon, the XSophe interface and the computational program Sophe is a state of the art package for the simulation of electron paramagnetic resonance spectra. The Sophe program performs the computer simulation and includes a number of new technologies including; the SOPHE partition and interpolation schemes, a field segmentation algorithm, homotopy, parallelisation and spectral optimisation. The SOPHE partition and interpolation scheme along with a field segmentation algorithm greatly increases the speed of simulations for most systems. Multidimensional homotopy provides an efficient method for accurately tracing energy levels and hence tracing transitions in the presence of energy level anticrossings and looping transitions and allowing computer simulations in frequency space. Recent enhancements to Sophe include the generalised treatment of distributions of orientational parameters, termed the mosaic misorientation linewidth model and a faster more efficient algorithm for the calculation of resonant field positions and transition probabilities. For complex systems the parallelisation enables the simulation of these systems on a parallel computer and the optimisation algorithms in the suite provide the experimentalist with the possibility of finding the spin Hamiltonian parameters in a systematic manner rather than a trial-and-error process. The XSophe software suite has been used to simulate multifrequency EPR spectra (200 MHz to 6 00 GHz) from isolated spin systems (S > ~½) and coupled centres (Si, Sj _> I/2).

Griffin, M.; Muys, A.; Noble, C.; Wang, D.; Eldershaw, C.; Gates, K.E.; Burrage, K.; Hanson, G.R."XSophe, a Computer Simulation Software Suite for the Analysis of Electron Paramagnetic Resonance Spectra", 1999, Mol. Phys. Rep., 26, 60-84.


Progress toward the structure determination of the nickel carbon monoxide dehydrogenase component of the acetyl coenzyme-A decarbonylase

synthase complex from M. barkeri

Bing Hao ~, Weimin Gong ~, Tom Tallant b, D.J. Ferguson b, Joseph A Krzycki b, and Michael K Chan ~. a Department of Biochemistry and Chemistry, The Ohio State University, 484 West 12 th

Ave, Columbus, OH 43210, USA (e-mail: [email protected]) b Department of Microbiology, The Ohio State University, 492 West 12 th Ave. Columbus, OH

43210, USA

Species of Methanosarcina are methanogens capable of growing on acetate as the sole source of carbon and energy. In M. barkeri, the acetyl coenzyme-A decarbonylase synthase (ACDS) complex plays a central role in acetyl-CoA metabolism. This multi-subunit complex has attracted considerable attention not only due to its fundamental biological functions in methanogenesis, but also because two of the last known nickel-containing cofactors within this complex remain to be structurally characterized. According to the specific activities of the ACDS complex, it can be divided into three components. The carbon monoxide dehydrogenase (NiCODH) component of the ACDS complex has an c~2s2 structure with unusual nickel-iron sufur clusters and catalyzes the reversible oxidation of CO to C02. In order to understand the mechanism of acetyl-CoA synthesis/degradation in methanogens, and to elucidate the structure of the nickel-iron sulfur cluster, we are working to determine the x-ray crystal structure of the c~2s2 NiCODH core of the ACDS complex. The current progress of the structure determination will be presented.

Spectroscopical Studies of Cytochrome cs, some exhibiting HALS EPR signals

E. Harbitz a, G. Zoppellaro a, T. Teschner b, S. Fauchald a, B. Katterle a, V. Schfinemann b, A. X. Trautwein b, D. Arciero c, A. Hooper c, S. Ciurli d, K. K. Andersson a "Department of Biochemistry, University of Oslo, Box 1041 Blindern, N-0316 Oslo, Norway blnstitute of Physics, Medical University of Liibeck, 23538 Liibeck, Germany CDepartment of Biochemistry, Molecular Biology and Biophysics, Univ. of Minnesota St.Paul, Minnesota 55108, USA

Department of Agro-Environmental Science and Technology, University of Bologna, 10 1-40127 Bologna, Italy.

An unusual low-spin cytochrome c-554 purified from the methane oxidizing bacteria Methylosinus Trichosporium OB3b, exhibits an HALS (highly axial low-spin) EPR signal when subjected to low temperature EPR spectroscopy. The spectrum has a typical HALS lineshape with a g-max value of 3.37 at pH 8.2 and pH 7.0 and small amounts of C J +. The EPR spectrum changes slightly with pH. MALDI-TOF Mass Spectroscopy has shown that this cytochrome has a molecular weight of 12 234 Da, and that it only contains one heme group. The CD spectrum of the oxidized form of this cytochrome shows a valley peak at around 710 nm, indicating an His / Met coordination of the heine iron. This cytochrome is compared with cytochrome cs purified from Methylocoeeus capsulatus (Bath), cytochrome c-552 form Nitrosomonas europaea which exhibits an HALS EPR signal I and has known 3D structure 2 and cytochrome c-553 from Bacillus pasteurifl. M6ssbauer data obtained at 4.2K in fields up to 7 T of N. europaea cytochrome c-552 have been analyzed by the method of Oosterhuis and Lang in full accord with the HALS EPR signal. Even if the c-type cytochromes have similar His / Met coordination of the heme iron they can exhibit an HALS EPR signal. The HALS signal could indicate a difference in the electron transport pathway to and from the heme group to other groups.

1. Arciero D., Peng Q. Y., Peterson J., A. FEBS Lett., 342,217-220 (1994) 2. Yimkovich R., Bergmann D., Arciero D., Hooper A., Biophys. J., 75, 1964-1972 (1998) 3. Benini S., Gonzfilez A., W. R. Rypniewski, K. Wilson, J.J. Van Beeumen, S. Ciurli, Biochemistry, 39, 13115-13126

(2000)


Streptonigrin analogues as potential anticancer agents

M a r g a r e t M. H a r d i n g a nd P ia I. A n d e r b e r g

School of Chemistry, University o f Sydney, NSW 2006, AUSTRALIA

Streptonigrin is a functionalized 7-aminoqumoline-5,8-dione that is highly active against a range of human cancers. The drug causes DNA strand scission in a process that involves transition metal ions and oxygen ~. All of the research that identified the crucial role of metal ions in the mechanism of antitumor action of streptonigrin was carried out after clinical trials ceased in 1977. Hence metal complexes of the drug were never administered during clinical trials. Targeting of metal complexes of streptonigrin as potential therapeutics may now be investigated as the structures and stabilities of several metal complexes has been established ~. Streptonigrin analogues containing the redox active quinolinequinone unit and the 2,2'-bipyridyl metal binding site have been prepared 2 and their interactions with transition metal ions studied by cyclic voltammetry,

O

CH30~y"~

- H2N,,~CH3

OCH 3

NMR spectroscopy and ultra-violet spectrophotometry. Studies of DNA binding and cleavage, oligonucleotide binding and topoisomerase II inhibition assays have clarified the role of the phenol ring D of streptonigrin in the mechanism of action and have shown the significant effect of metal ions in modulating the redox potential of the quinone.

1. Harding, M. M. and Long, G. V., Current Medicinal Chemistry, 4, 405 - 420 (1997) 2. Kimber, M. C., Anderberg, P. I. and Harding, M. M., Tetrahedron. 56, 3575 - 3581 (2000)

New Ferrocene-substituted Glucose Compounds

Chr is t i an G. H a r t i n g e r a, A l e x e y A. N a z a r o v a, V l a d i m i r B. A r i o n a, Ge ra ld Gies te r b, M i c h a e l J a k u p e c a,

M a r k u s Ga lansk i a and B e r n h a r d K. K e p p l e r a

"Insti tute of Inorganic Chemistry, University of Vienna, Waehringer Str. 42, A-1090 Vienna, Austria, (e-mail: Christian. [email protected]. at) Institute of Mineralogy and Crystallography, University of Vienna, Althanstr. I4, A-1090 Vienna, Austria

Monosaccharides belong to the basic biomolecules as they are the building blocks for some of the most important biological compounds: polysaccharides, nucleic acids and glycoproteins.

Coordination compounds of carbohydrates are taking part in biological processes. Several transition metal complexes have been synthesised, characterized and also tested in vitro as well as in vivo to be used in cancer therapy ~. One of the problems of metal complexes in cancer therapy is their toxicity 2.

We report here about the synthesis and characterization of compounds containing one or two molecules of 1,1 "-dicarbonylferrocene on O-methyl-c~-D- glucopyranoside or glucosamine. The absolute configuration of 2,3-(ferrocene-

CII8! Ctl91

~171 , CIISl 571 ~81 0t5~

CI41 Cl CISI 01~

1,1 '-dicarbonyl)-O-methyl-(x-D-glucopyranoside has been determined by single crystal X-ray diffraction analysis. First in vitro tests of our compounds showed low cytotoxicity on four human tumor cell lines. We expect this type of molecules to be an interesting precusor for antitumor agents with lower toxicity and higher activity.

1. Wong E., Giandomenico C. M., Chem. Rev., 99 (9), 2451 - 2466 (1999) 2. Yano Shigenobu, Coord. Chem. Rev., 92, 113 - 156 (1988)

We gratefully acknowledge the FWF (Austrian Science Fund) for financing the project (Project Number: P-12381).

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