abstracts poster presentations (q)

Journal of Inorganic Biochemistry 86 (2001) 281

Electron-transfer through biological 'wires' in multi-centre enzymes

Lars J.C. Jeuken a, Anne K. Jones a, Stephen K. C h a p m a n b, Gary Cecchini c, Fraser A. Armstrong a a Inorganic Chemistry Laboratory, Oxford University, South Park Road, OX1 3QR, Oxford, U.K.

(e-mail: Lars.Jeuken@chem.ox.ac. uk) b Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road,

EH9 3J J, Edinburgh, U.K. c DVA Medical Center, University of California, CA 94121, San Francisco, U.S.A.

The multi-centre electron-transfer reactions of two fumarate reductases has been studied by cyclic (CV) and square-ware voltammetry (SWV). The two enzymes from S. frigidimarina (Fcc3) and E. coli (Frd, soluble domain) were adsorbed on an electrode surface ('edge' oriented pyrolytic graphite) to avoid complications due to protein diffusion and, therefore, to focus on the fast and direct electron transfer (ET) between the electrode and the active sites. Both FCC 3 and Frd contain a buried active-site flavin group (FAD), which is 'connected' to the surface of the protein by biological 'wires'. Since the FAD is a cooperative two-electron transfer, the signal due to the FAD redox transitions is clearly distinguished above those due to the centres of the 'wire'. These

i. ~-~,7 r.7,

'wires' consist, respectively, of four hemes (Fcc3) or three Fe-S clusters (Frd, see Figure). By using large values (0.075 - 0.3 V) for the square-wave amplitude, the electron-transfer rate can be measured over a

continuously variable driving force in either direction. In combination with previous work on smaller a one-electron transfer protein, the intra-molecular ET transfer between the electrode and the buried FAD group could be studied. The SWV data have been simulated using a range of models which represent possible ET mechanisms. While the data of Frd suggest that (in part) the electrons 'hop' from electrode to 'wire' and from 'wire' to the FAD, the Fcc3 data indicate that the electrons can transfer directly to the active site, suggesting that there is also a super-exchange mechanism through the hemes.

Hybrid density functional (DFT) approach to the tH and 14N hyperfine tensors in the blue copper proteins

Julia Jezierska, Adrian R. Jaszewski Faculty of Chemistry, University of Wroclaw, (e-mail: julia@wchuwr, chem. uni. wroc.pl)

14 Joliot-Curie St., 50-383 Wroclaw, Poland

Recent advances in the ENDOR, ESEEM techniques allowed for study of the hyperfine interactions with ligands nuclei in the copper site of blue copper proteins 1. The aim of this work is to resolve the problem of some uncertainty in assignment of the hyperfine splittings to the remote nitrogen and hydrogen nuclei by comparison with the hyperfme couplings obtained directly from quantum chemical calculations. It has been shown, also by us 2, that DFT satisfactorily reproduces EPR parameters, although the calculations need to use the method providing an accurate description of electron correlation and basis sets properly describing core and valence regions with the outer core-inner valence region of sufficient flexibility. We report UB1LYP hybrid density functional studies on the hyperfine parameters of wild-type azurin from Pseudomonas aeruginosa and MI 21Q mutant of azurin from Alcaligenes denitrificans. The large models of copper sites used in the calculations give quantitative insight into the spin density distribution and confirm highly delocalized character of the unpaired electron. Theoretically predicted values of isotropic and anisotropic hyperfine couplings and their relation permit to verify the assignment of e.g 14N hyperfine splittings to the coordinated and remote nitrogens of His117 and His46, to the backbone nitrogens of Asn47 (previously assigned to Cys112), Thr113 and Cys112 (the smallest hfs values) - in the wild-type azurin.

1. M.M. Werst, C.E. Davoust and B.M. Hoffman, J.Am.Chem.Soc. 113, 1533(1991); J.W.A. Coremans, M. van Gastel, O.G. Poluektov, E.J.J. Groenen et al. Chem.Phys.Lett. 235,202(1995); M. van Gastel, J.W.A. Coremans, L.J.C. Jeuken, G.W. Canters and E.J.J. Groenen, J.Pbys.Chem. A 102, 4462(1998) and references therein

2. A.Jaszewski, J.Jezierska et al. Chem.Phys.Lett.,319, 611(2000); ibid.,331,403(2000); ibid.,340, 581(2001), 3. ibid.,341,168(2001)

282 Journal of Inorganic Biochemistry 86 (2001)

15N N M R mobility studies on rusticyanin: elucidating the factors that determine the high stability of this blue copper protein at low pH's

Beatriz Jim6nez a, Antonio Donaire a, Jos6-Maria Moratal b, Mario Piccioli ~, John F. Hall °, and S. Samar Hasnain dx.

Departamento de Ciencias Quimicas, Universidad Cardenal Herrera-CEU, Avda. Seminario s/n, 46113-Montcada, Valencia, SPAIN (e-mail: beatriz@uch.ceu.es) b Departamento de Quimica Inorgfnica. Universitat de Valbncia. C/Dr. Moliner, 50. 46100- Burjassot. Valencia. SPAIN

Magnetic Resonance Center, University of Florence, Via L. Sacconi, 6, 50019 Sesto Fiorentino, ITAL Y d The Cell Signalling Laboratory, Department of Biological Sciences, De Monfort University, The Gateway, Leicester LE1 9BH, U.K.

CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD, U.K.

Rusticyanin (Rc) is a Blue Copper Protein (BCP) very stable in a large range o fpH values (1.5-10). The overall folding of this protein consist of a [3-barrel structure and two c~-helices. Since this is the typical structure of BCPs, and these proteins do not show stability for pH values lower than 4.0, other secondary and tertiary contacts must account for the high stability of Rc. Moreover the mobility of each aminoacid in different time scales can proportionate clues for elucidating the essential factors in the stability of the protein. We have determined the relaxation rates (T~ and T2) of the ~SN of the amide backbone nuclei, and the ~H-1~4 NOE values at two different magnetic fields (500 and 600 MHz). Then, we have performed a model free analysis with these dat#. We can conclude that most of the peptidic nitrogen nuclei have a behaviour that correspond to the model 1 or model 2 kinds (without exchange processes). Regions with different type of motion can be related to less stable parts of the protein.

Mandel A.M., Akke M. and Palmer A.G., J. Mol. Biol., 246, 144-163 (1995).

Structural and functional studies of rubrerythrin from Desulfovibrio vulgaris

Shi Jin a, Zhi-Jie Liu b, John Rose b, Bi-Cheng Wang b, Donald M. Kurtz, Jr. a Department of Chemistry, University of Georgia, Athens, GA30602, USA

(e-mail: sjin@chem, uga. edu) b Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA30602, USA

Rubrerythrin (Rbr), found in anaerobic or microaerophilic bacteria and archaea, is a non-heme iron protein containing an oxo-bridged diiron site and a rubredoxin-like [Fe(SCys)4] site. The physiological role of Rbr has not been conclusively established. Evidence that Rbr is a novel NADH peroxidase has been published.l'2 The diferrous site in reduced Rbr was assumed to react with the substrate hydrogen peroxide with the electrons transferred from the Fe(II)S4 site through a "head-to-tail" dimer interface. Only the structure of the fully oxidized (all-Fe 3÷) Rbr has been reported. 3 In order to investigate the molecular mechanism of the NADH peroxidase reaction, both the reduced form of Rbr and its azide adduct were crystallized, and their structures were determined by X-ray crystallography using difference Fourier analysis. In comparison to the oxidized Rbr, the structure of reduced Rbr supports a H56/E97 ligand-exchange mechanism. The structure of reduced Rhr azide adduct may mimic a substrate-binding mode. The reduction of H202 by the reduced Rbr was investigated by stopped-flow spectrophotometry. Current results indicate that each Rbr diferrous site gives two electrons to I-I202 through two weak bonds that were formed rapidly between the substrate and two solvent ligands.

• -%

1. Lumppio H., Shenvi N., Summers A., Voordouw G., Kurtz D. M. Jr., Journal of Bacteriology, 183, 101-108 (2001) 2. Coulter E., Shenvi N., Beharry Z., Smith J., Prickril B., Kurtz D. M. Jr., Inorganica Chimica Acta, 297, 231-241 (2000) 3. DeMar6 F., Kurtz D. M. Jr., Nordlund P., Nature Structural Biology, 3,539-546 (1996)

Journal of Inorganic Biochemistry 86 (2001) 283

Mult i funct ional conjugates for f luorescence and magnet ic resonance imaging

Br ian P. J o h n s o n a, A n d r e w J. E w a l d b, and T h o m a s J. M e a d e b

~ Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 127- 72, Pasadena, CA 91125 USA (e-mail: brianj@caltech.edu)

b Division of Biology, California Institute of Technology, Pasadena, CA 91125 USA

The investigation of cell lineage and patterning of the central nervous system is an active area of research. Computer-enhanced light microscopy imaging and magnetic resonance imaging (MRI) have emerged as leading techniques for in vivo monitoring of intact organisms. Employing the advantages of both techniques, a set of bifunctional polymeric imaging agents is described. A high-molecular weight, membrane impermeable scaffold such as dextran was chosen, so that the imaging agent would be trapped inside cells after injection. The molecular framework is covalently functionalized with multiple complexes of both gadolinium-diethylenetriammepentaacetic acid (DTPA) and a fluorescent dye, such as the lipophilic 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). A ~picat 65mer of dextran (10.5 kD tool. weight) is modified with as many as 15 Gd-DTPA units and 3 to 5 DiI molecules, enough to promote membrane affinity for this inherently hydrophilic polymer system. The synthesis of this class of reporter probes is versatile, allowing for varying wavelengths of attached fluorophores and types of MR agents to be ased

Insights into the catalytic cycle of [NiFe]-hydrogenases from direct e lectrochemistry

Anne K. Jones a, Harsh R. Pershad a, Emma Sillery a, Simon P. J. Albracht b, Fraser A. Armstrong a " Inorganic Chemistry Laboratory, Oxford University, South Parks Road, OX1 3QR, Oxford,

United Kingdom (e-mail.- anne.jones@icl.ox.ac.uk) /' Swammerdam Institute for Life Sciences, University of Amsterdam, Plantage Muidergracht 12,

NL-IO18 TV, Amsterdam, The Netherlands

We have used protein film voltammetry (PFV) ofAllochromatium vinosum (Av) UNiFe]-hydrogenase to address the question: what factors control the rate of catalysis of a complex electron transport enzyme? PFV is a direct electrochemical technique that obtains thermodynamic and kinetic data from an enzyme layer adsorbed on an electrode, an arrangement analogous to the interaction of an enzyme with its natural redox partner. Previous work showed that hydrogen oxidation by this enzyme layer is faster than with redox mediators ~. For enzymes adsorbed at an electrode, rate determining steps could include mass transport of substrate, binding/dissociation of substrate/product, electron transfer to/from substrate, intramolecular electron transfer within the enzyme, and interfacial electron transfer with the electrode (redox partner). We now show that the observed rate and rate limiting step of Av [NiFe]-hydrogenase depend on the conditions employed in the experiment. We demonstrate voltammetry both under mass transport controlled conditions and under enzymatic control and interpret these data mechanistically.

1. Pershad, H. R., Duff, J. L. C., Heering, H. A., Duin, E. C., Albracht, S. P. J., and Armstrong, F. A., Biochemistry, 38, 8992-8999, 1999.

We acknowledge the Rhodes Trust, UK EPSRC, BBSRC, NWO and SON for financial support.

284 Journal of Inorganic Biochemistry 86 (2001)

Construction, overexpression, and purification ofArthrobacter globiformis amine oxidase Strep-tag II fusion protein.

Gregory_ A. Juda, David M. Dooley Department of Chemistry and Biochemistry, Montana State University 5971 7, Bozeman, USA (e-mail. gjuda@hotmail, com)

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS media. For example, we have demonstrated that 50 mg of protein can be obtained in two days from 2 L of E. coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.

1. Juda G.A., Bollinger J.A., and Dooley D.M., Protein Expression and Purification, in press.

Kinetic studies on the copper(II)-mediated oxygenolysis of the flavonolate ligand processing via a 1,2-dioxetane intermediate

J6zsefKaizer a and G~bor Speier b

aResearch Group for Petrochemistry, Hungarian Academy of Sciences, 8201 Veszpr~m,HUNGARY (e-mail. kaizer@almos.vei, hu)

bDepartment of Organic Chemistry, University of Veszpr~m, 8201 VeszprOm, HUNGARY

The degradation of flavonols in living systems to the corresponding depsides (phenolic carboxylic esters) and CO are performed by flavonol 2,4-dioxygenase. From our earlier results obtained with redox and non-redox metal- containing model systems, the conclusion can be drawn that the oxygenolysis of the flavonolate ligand in aprotic solvents takes place via an endoperoxide intermediate 1-3.

[Cu(L)(4'R-fla)2 ] (L = phen, bpy, tmeda; R = H, OCH3, CH3, CI) complexes have been prepared by reacting [Cu(4'R-fla)2 ] complexes with the nitrogen-containing coligands. Oxygenation of these complexes results in the corresponding (O-benzoylphenylglyoxylato)copper complexes [Cu(L)(O-bphga)2] via 1,2-dioxetane intermediates. The oxygenation of [Cu(phen)(fla)2 ] shows chemiluminescence and in the emission spectrum bands at 506, 546 and 578 nm

were found assigned to the decomposition of the 1,2-dioxetane species. Labelling experiments with 1802 evidenced the

incorporation of both 180 atoms of |802 into the O-bphga ligand. The oxygenolysis of the flavonolato complexes follow the rate law -d[Cu(L)(4'R-fla)2]/dt = k[Cu(L)(4'R-fla)2][O2]. The reaction fits a Hammett linear free energy relationship for 4'-substituted flavonolates, and an increase of the electron density on copper makes the oxygenation reaction faster.

1.Balogh-Hergovich, 1~., Kaizer, J., Speier, G., Fiil6p, V. and Pfirkfinyi, L., Inorg. Chem., 38, 3787-3795 (1999) 2.Barhhcs, L, Kaizer, J. and Speier, G., J. Org. Chem., 65, 3449-3452 (2000) 3.Balogh-Hergovich, I~., Kaizer, J., Speier, G., Huttner, G. and Jacobi, A., Inorg. Chem., 39, 4224-4229 (2000)

We thank the Hungarian Research Fund (OTKA T 030400) for support.

Journal of Inorganic Biochemistry 86 (2001) 285

Reversible O2-binding of ~Hq2:r12-peroxodicopper(II) complexes: The O2-affinity control with the distortion of the coordination geometry around Cu(II) ions

Yuji Kajita, Shinsuke Tanaka, Masahito Kodera, and Koji Kano. Department of Molecular Science and Technology, Doshisha University, Kyotanabe Kyoto, 610-

032],Japan (e-mail: mkodera@mail, doshisha.ac.jp)

Hemocyanins (Hc, arthropodal and molluscan blood O2-carrier proteins) bind 02- molecule reversibly. We reported crystal structure and reversible Oz-binding of a room temperature stable ~-rl2:lqZ-peroxodicopper(II) complex [Cuz(H6Mh)(Oz)](PF6)z (1) (H6Mh = 1,2-bis[2-(bis(6-methyl-2-pyridyl)methyl)-6-pyridyl]ethane).! On the basis of the fact that the square pyramidal geometry of Cu(II) ions in 1 is largely distorted, it was suggested that the distortion is one of the important aspects to control the reversible O2- binding of the l,t-qZ:qZ-peroxodicopper(II) complex. Here, we synthesized new hexapyridme ligands M6Mh and mH6iPMh to clarify if the distortion of the copper coordination geometry i,s responsible for the reversible O2-binding. The dicopper(I)

R' R' H 6 M h : R = H , R ' = R " = M e

M 6 M h : R = R ' = R " = M e m H 6 [ P M h : R = H , R ' = i P r ,

R " = M e

complex of M6Mh [Cuz(M6Mh)(MeCN)z](PF6)2 (2) binds O2-molecule in CHzCI2 at -20 ZC to form the ~-r[2:]] 2- peroxodicopper(II) complex [Cu2(M6Mh)(Oz)](PF6)z (3). The UV-vis spectrum of 3 shows two absorption bands at 366 (21000 M-~cm -1) and 537 nm (2000 M-lcmt). The resonance Raman spectrum of 3 shows a Vo-o band at 765 cm ~ (724 cm -! with !8Oz). 3 releases Q-molecule in MeCN-CHzC12 at 50 •C and is regenerated under 02 at -20 ~C. The 02- releasing temperature (50 i~ C) for 3 is much lower than 80 I-C for 1. Since the copper coordination geometry of 3 is more distorted than 1, it seems that the la-qz:rlZ-peroxodicopper(II) complex releases O2-molecule more easily with the increasee of the distortion of the copper coordination geometry.

1. M. Kodera, et. al. J. Am. Chem. Soc. 1999, 121, 11006.

Effect of detergents on the purification of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Toshiaki Kamachi, Akimitsu Miyaji, Ichiro Okura Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku,

Yokohama, 226-8501, Japan (e-mail: tkamachi@bio.titech.ac.jp)

Methane monooxygenase (MMO) catalyzes the hydroxylation of methane to methanol. Though particulate MMO (pMMO) has been purified by the several groups, the enzymatic activities were low. The effect of detergents on the purification of pMMO from Methylosinus trichosporium OB3b and the pMMO activity was carried out. Among the detergents tested dodecyl-13,D-maltoside was the best for the solubilization of the pMMO from membrane, pMMO activity was concomitantly increased by the removal of dodecyl-lg,D-maltoside from the pMMO solution and decreased by the addition of dodecy-[3,D-maltoside. These data indicate that the dodecyl-[3,D-maltoside inhibits the pMMO reversibly. After solubilizing the pMMO from membrane, pMMO was purified by ionic exchange column chromatography using Brij 58 as a detergent. The purified pMMO exhibits three subunits (43 kDa, 27 kDa, 24 kDa) on the SDS-polyacrylamide gel electrophoresis. These molecular weights are consistent with the data reported previously. Specific activity for the purified pMMO was 0.87 nmol rain 1 mg -1, indicating the purified pMMO retains high activity. The metal analysis of the purified pMMO reveals that pMMO contains only copper ions but no iron ions. The EPR spectrum of the purified pMMO also supports the existence of copper ions in the molecule as shown in figure. The EPR signals found in the pMMO is typical type II copper (II) and there is no iron signal around g=4.0-6.0.

I I I I ! 100 200 300 400 500

Magnet ic field / mT

Figure X-band ESR spectrum of purified pMMO from M. trichosporium OB3b.

286 Journal of lnorganic Biochemistry 86 (2001)

Character izat ion of a copper chaperone, MelC1 from Streptomyces glaucescens

Ejan M. Kamlin, Kasey Konesky, David M. Dooley Department of Chemistry and Biochemistry, Montana State University 5971 7, Bozeman, USA (e-mail: ejanl9@hotmail, com)

The melanin operon (MelC) of Streptomyces glaucescens contains two genes, MelC1 and MelC2. MelC2 is the structural gene for tyrosinase (apotyrosinase), while the function of MelC1 is to facilitate copper incorporation into tyrosinase via a binary complex between the two proteins. Streptornyces glaucescens MelC1 has been expressed in Streptomyces lividans TK24 as a Streptag-II fusion protein allowing for its efficient purification. In order to better understand the transactivation of tyrosinase, a full characterization of MeIC 1 is imperative.

C y c l i c - v o l t a m m e t r i c study on complexat ion of anionic (porphinato) iron with per- -methylated-[3-cyclodextrin

Kqji Kano, Yumiko Sone Department of Molecular Science and Technology, Doshisha University, Kyotanabe, Kyoto 610- 0321, Japan (e-mail.kkano@mail.doshisha.ac.jp)

[5,10,15,20-Tetrakis(4-sulfonatophenyl)porphinato]iron(III) (FeTPPS) is known to form 1:1 and 1:2 complexes

with (2,3,6-tri-O-methyl)-13-cyclodextrin (TMe-]3-CD) at low and high pH ranges, respectively. Such a phenomenon

suggests a possibility of a pH-responsive molecular switch. In the present study, we checked the molecular switching by

cyclic voltammetry. In the absence of cyclodextrin, FeTPPS in 0.1 M

aqueous Na2SO4 at pH 2.0 (HC104) showed the reduction potentials (Ep red) at -248 (Fe 3+ to Fe 2+) and -1126 mV (Fe z+ to Fe+). The oxidation potential (Ep

o~) was observed at -166 mV (Fe z+ to Fe3~). Meanwhile, the Ep r~a value for Fe 3. to Fe z+ and the Ep ox value for Fe z+ to Fe 3+ were -240 and 544 mV,

respectively (Fig. 1). The AEp ox value is ca. 700 mV, though the shift in Ep

~d is not so remarkable. The redox reactions such as Fe 2+ = Fe ~ and Por =

Por "+ were not observed in the presence of TMe-[3-CD. At pH 2.0,

Fe(III)TPPS exists as a diaqo form (Fe(III)TPPS(H20)2). Under such

conditions, the net charge on the porphyrin ring is +1 where a 1:1 complex of

FeTPPS and TMe-[3-CD is formed mainly. The 1:1 complex may be reduced

electrochemically to Fe(II)TPPS(H20)2 whose net charge on the porphyrin ring

is zero. Fe(II)TPPS(H20)2 forms a 1:2 complex whose metal center is strictly

covered by two CD cavities. Therefore, the oxidation of this complex seems to

hardly occur on the electrode.

.32~ !

-24~ .C1~ ~22: ..... Z1F -~3: -8 ~ / f fp~= m i

+32J i r I

+4oi -1.2 -0.8 -0.4 0.0 " +0.4 ' +0,B

P o t e n t i a l / V (vs. Ag/AgCI)

Fig. i. Cyclic voltammogram of I mM FeTPPS in 0.1 M aq. Na2SOa in presence of2mM TMe-~-CD at pH 2.0.

Journal of Inorganic Biochemistry 86 (2001) 287

Quantification of the steric effect of the (C6)NH 2 group on metal-ion binding at N1 of the adenine residue

Lar i sa E. K a p i n o s , H e l m u t S ige l

Institute of Inorganic Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland (e-mail. helmut.sigel@unibas.ch)

The adenine residue is an important potential binding site for metal ions in their interaction with nucleotides or nucleic acids. The N3 atom coordinates to metal ions only under very special conditions; l,x the prime binding sites are

NH2 NI and N7. Both sites, N1 and N7, are under the steric influence of the (C6)NH 2 group which itself does

~/~a~//.jN 1 not bind metal ions. The steric effect of this amino calculated for ligands with p / ~ = 4.00

group on metal ion binding at N7 was quantified M2 + log K~Mfey ) log /~M(oA-Py) log A recently 3 via complexes of benzimidazole Ca 2+

! adenine derivatives'4 Now we are attempting the -0.08 _+ 0.07 -0.13 +_ 0.13 -0.05 + 0.15 R res idue corresponding quantification for the N1 site. To this Mg 2+0'04 -+ 0.04 -0.06 +_ 0.06 -0.10 + 0.07

end we determined the log ~ e versus p / ~ straight-line plots for Mn2+0.29 -+ 0.02 -0.02_+ 0.07 -0.31 + 0.07 Cu 2+ 2.05 + 0.04 0.70 + 0.07 -1.35 + 0.08 several metal ions with six pyridine-type ligands (Py) which had no - - -

ortho substituents and with five derivatives which had amino or methyl Zn2+ 0.80 + 0.02 0.01 +_ 0.08 -0.79 +_ 0.08 groups in the ortho position (oA-Py); tubercidin (7-deazaadenine) was one of these latter ligands. With the resulting straight line equations for any known pK a value of a ligand the expected complex stability can be calculated. A few results are summarized in the Table for PKHHL = 4.00, which is close to the pK a value of the adenine residue. The varying steric effect of the (C6)NH 2 group is evident.

1. Meiser C., Song B., Freisinger E., Peilert M., Sigel H. and Lippert B., Chem. Eur. J., 3,388-398 (1997) 2 Blindauer C.A., Emwas A.H., Ho13)A., Dvo~fikovh H., Sletten E. and Sigel H., Chem. Eur. J., 13, 1526-1536 (1997) 3 Kapinos L.E., Ho13) A., Gtinter J. and Sigel H., Inorg. Chem., 40, in press (2001) 4. Kapinos L.E., Song B. and Sigel H., Chem. Eur. J., 5, 1794-1802 (1999) Supported by the Swiss Nat. Science Foundation and within COST D20 by the Swiss Fed. Office for Education & Science.

Structure of benzoate dioxygenase reductase from Acinetobacter sp.

A. K a r l s s o n a, Z. B e h a r r y b, K. L e e c, S. R a m a s w a m y d, D. Kur t z b, H. E k l u n d a

Department of Molecular Biology, Swedish University of Agricultural Sciences, BMC Box 590, S-75124, Uppsala, Sweden,(e-mail andreas@xray.bmc.uu.se)

~' Department of Chemistry, University of Georgia, 30602-2556, Athens, Georgia, USA. c Department of Microbiology, Changwon National University, Changwon-si, Kyongnam, 641-

773, S. Korea. C Department of Biochemistry, University of Iowa,IA 52242-1109, lowaCity, Iowa USA.

The system for degrading benzoate by benzoate dioxygenase is similar to the degradation of Naphthalene by naphthalene dioxygenase. To complete de dioxygenetion there are two electrons required in both cases. In the case of the Naphthalene dioxygenase system the electrons are transferred from NADPH via a reductase and a ferredoxin to the terminal dioxygenase component. However in the case of benzoate dioxygenase the electron transfer is mediated by a single protein, the benzoate dioxygenase reductase, encoded by the BenC gene in Acinetobacter sp. This protein has been cloned, overexpressed and purified by Z. Beharry & D. Kurtz, our collaborators in Athens, Georgia. Crystals of the reductase component has been grown in our lab. Native data has been collected as well as data on heavy metal derivatives. The structure was solved using the MAD method and refined at 1.50 A resolution. It consists of three distinct domains; one binds N A D H , the second a FAD-cofactor and the third a [2Fe2S] cluster. We intend to solve the structure of the reductase with bound NADH to further examine the electron transfer in the system.

288 Journal of lnorganic Biochemistry 86 (2001)

Conformation, recognition by HMG-domain proteins and nucleotide excision repair of DNA monofunctional adducts of novel antitumor bifunctional platinum

complex trans-[PtCl2(NH3)(thiazole)]

Jana K a s p a r k o v a a, N i c h o l a s Farre l l b, V i k t o r B r a b e c a

" Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ- 61265 Brno, CZECH REPUBLIC (e-mail. j a n a @ i b p . c z ) b Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284- 2006, USA

Recent findings that novel trans-dichloroplatinum(II) complexes exhibit antitumor activity violate the classical structure- activity relationships of platinum(II) complexes. These novel "nonclassical" trans-platinum complexes also comprise those containing planar aromatic amines. The initial studies have shown that these compounds form DNA interstrand cross-links (up to ~30 %) and considerable amount of adducts remains monofunctional. The present work has shown that major monofunctional DNA adducts of trans-[PtC12(NH3)(thiazole)], a representative of the group of new antitumor trans-dichloroplatinum complexes containing planar amines, create a local conformational distortion, which includes a stable curvature (34 ° towards major groove), unwinding (13 °) and the distortion is localized to 5 base pairs around the adduct. Hence, this distortion is very similar to that produced in DNA by major 1,2-GG intrastrand cross-link of antitumor cisplatin. In addition, we have observed recognition of these monofunctional adducts by HMG1 proteins, but we have observed effective removal of these adducts from DNA by nucleofide excision repair. These results suggest that the processing of the monofunctional DNA adducts of trans-[PtClz(NH3)(thiazole)] in tumor cells may be similar to that of the major adducts of"classical" cisplatin. The results further support the view that the simple chemical modification of

structure of an inactive compound alters its DNA binding mode into a DNA distortion of an active drug.

This research was supported by the Grant Agency of the CR (grant no. 305/99/0695) and the Internal Grant Agency of the Ministry of Health of the CR (grant no. NL6058-3/2000).

Spectroscopic and electrochemical properties of the Metl50Gln mutant of acromobacter cycloclastes nitrite reductase

Kunishige Kataoka a, Shinobu Sakai b, Kazuya Yamaguchi b, and Shinnichiro Suzuki b a Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, JAPAN (e-mail: kataoka@cacheibm.s.kanazawa-u.ac.jp) b Department of Chemistry, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043, JAPAN

The copper-containing nitrite reductase from Achromobacter eycloclastes (AcNIR) has two types of Cu centers; type 1 (two His, Cys, and Met ligands) and type 2 (three His and solvent ligands) coppers. Type 1 Cu receives one electron from pseudoazurin (pAz) and transfer it to the reaction center, type 2 Cu, catalyzing one electron reduction of nitrite ion to nitric oxide. The mutant replacing the Metl50 with Gln has been prepared and characterized to investigate the axial methionine ligands' effect on the reduction potential, spectroscopic and electron transfer properties of type 1 Cu center of AcNIR. The mutant (M150Q-AcNIR) shows the electronic absorption and CD spectra considerably similar to those of blue Cu proteins mavicyanin (Mv) and stellacyanin (St) having Gln ligand. The EPR signal of type 1 Cu of the mutant has an axial character with smaller A//value compared to that of wild-type enzyme. As the case of M121Q-azurin ~, M95Q-pAz 2, and M467Q mutant of bilirubin oxidase 3, the substitution of Metl50 to Gln may induce a more highly tetrahedral distortion of the copper site. Metl50Q-AcNIR retains about 80% nitrite reducing activities of wild-type AcNIR with benzylviologen as an electron donor. When reduced pAz was used as a donor, the mutant can not oxidize pAz (no detectable activity). These results indicate that the type 1 Cu of the mutant has negatively sifted midpoint potential. Therefore, intermolecular electron transfer between pAz and the mutant was interrupted.

1. Romeo A. et al. J. Mol. Biol., 229, 1007-1021 (1933) 2. Kataoka K., Kondo A., Yamaguchi Y., and Suzuki S., J. Inorg. Biochem., 82, 79-84 (2000) 3. Shimizu A. et al. J. Biochem., 152, 662-668 (1999)

Journal of Inorganic Biochemistry 86 (2001) 289

Blood glucose normalizing effect and action mechanism of vanadyl-phosphonate complexes

Kenji Kawabe, Hiromu Sakurai Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, 607-8414, Kyoto, JAPAN (e-mail: kkawabe@poppy.kyoto-phu.ac.jp)

Much interest has been focused on the potency of vanadyl (VO) complexes as an orally active antidiabetic agent. ~ Since the numbers and varieties of the insulinomimetic VO complexes reported to date are still limited, we prepared a new series of VO complexes with ligands containing phosphonate group.

In vitro insulinomimetic activity of each complex was higher than VOSO4 (VS) in terms of ICs0 value, 50% inhibition concentration of epinephrine-stimulated free fatty acids (FFA) release from isolated adipocytes. Such insulinomimetic activities of VS and VO complexes were partially inhibited by inhibitors of glucose transporter (GT) and several key enzymes on insulin transduction pathway, but not inhibited by insulin receptor (IR) and phosphatidylinositol 3-kinase (PI3K) inhibitors. These results indicate that VO compounds act at multiple sites including GT other than IR and PI3K in rat adipocytes. In in vivo trial, high blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats) were normalized within 7 days both after daily intraperitoneal and oral administrations of the complex. Thus, vanadyl- phosphonate complexes are proposed here to be a new candidate for orally active therapeutic of diabetes mellitus.

1. Sakurai H. and Tsuji A., in: Nriagu J.O. (Ed.) Vanadium in the Environment. Part 2: Health effects, John Wiley & Sons, New York, pp 297-315 (1998).

K.K. was supported by a postdoctoral fellowship from Kyoto Pharmaceutical University and has been supported by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

Effect of metal-assembling on Cu-mediated O2-oxidation of 3,5-di-tert-butylcatechol

Tomohisa Kawata, Masahito Kodera, and Koji Kano. a Department o f Molecular Science and Technology, Doshisha University, Kyotanabe Kyoto, 610-

0321,Japan (e-mail: mkodera@mail.doshisha.ac.jp)

Dinuclear copper(II) complexes, [Cu2(OH)2(TMED)2](C104) 2 (1), [Cu2(OH)2(TEED)2] i ~ _ ~ g . . . ~ (C104)2 (2), [Cu2(OH)2(EtC4ON4)](C104)2 (3) and tetranuclear copper(II) complexes, [Cu4(OH)4 (MeCnN4)2](C104)4 (n =4,(4), 5,(5) ,6,(6)) were synthesized. The crystal structures of 1 - 6 determined by X - ray crystal structure analyses. In the structure of 3, copper(II) ion has a distorted square planer geometry. In the structure of 4 - 6, two Cu2(~t-OH)2 cores end-capped by four en moieties of two MeCnN4 ligands are located at a distance by the -(CH2)- spacer as shown in the Figurel. The ESI mass spectra of 4 Figurel.ORTEP diagram of - 6 in MeCN show two peaks corresponding to {[CuE(OH)2(MeCnN4)](C104)} + and [Cu4(OH)4(MeC5N4)2](C104)4 {[Cu4(OH)4(MeCnN4)2](C104)3} +. In MeCN, 4 - 6 are in equilibrium between the di- and tetranuclear complexes and the former is the major species. 1 - 6 catalyze the O2-oxidation of 3,5-di-tert- butylcatechol (DTBC) to 3,5-di-tert-butyl-l,2-benzoquinone (DTBQ). The reactions were kinetically investigated. The pseudo lst-order rate constants obtained from the increase rates of DTBQ are proportional to 2nd-order with the catalysts and 1st-order with 02 for 3 - 6. The rate constants for 4 - 6 are 10-fold larger than that for 1 - 3. Formation of a mixed- valent Cu(I)Cu(II) species was shown by ESR spectra, and quantitative generation of H202 was detected by peroxyoxalate chemiluminescence reaction. The catalytic reactions start with a rapid two-electron oxidation of DTBC after formation of a tetranuclear Cu(II)4 - DTBC complex to give two molecules of the reduced dinuclear Cu(I)Cu(II) intermediate and DTBQ, followed by the rate-determining two-electron reduction of O2 by two Cu(I)Cu(II) intermediates to generate H2O 2. Because of the favorable tetranuclear structures, 4 - 6 can accelerate the rate-determining two-electron transfer from two Cu(I)Cu(II) intermediates to 02.

290 Journal of lnorganic Biochemistry 86 (2001)

Chiral lanthanide complexes

Suzanna Kean, a Gabriella Bobba, a C61ine Mathieu, a David Parker a and Martin Smith. a

"Department of Chemistry, The University of Durham, South Road, Durham, DH1 3LE, UK. (e-mail. s.d. kean@durham .ac. uk)

The lanthanide complexes of the previously reported chiral ligand 1 have been shown to exhibit very distinctive behaviour in their binding of [(CG6)], [(AT6)] and [CGCGAATTCGCG]2 through interrogation by ESMS, ~H-NMR, absorbance, difference circular dichroism, fluorescence quenching and Ln luminescence emission spectroscopy.~ The primary component of the free energy of binding has been linked to an intercalative interaction. The introduction of a chiral centre adjacent the N-methyl phenanthridinium chromophore in 1, imparts stereoconformationality to the antenna/intercalating moiety. It was envisaged that such a change would impart a greater difference in the binding behaviour of the resulting lanthanide complexes.

r[•e

RH,J o

. ~ { Ph Me NHR R Me.,.~/ p h,~,. /

Ligand 1

I G. Bobba, R.S. Dickens, S.D. Kean, C.E. Mathieu, D. Parker, R.D. Peacock, G. Siligardi, M. J. Smith, J.A.G. Williams, and C.F.G.C. Geraldes. J. Chem Soc., Perkin Trans. 2, 2001, in press.

We thank the EPSRC and BBSRC for grant support and the COST-D18 Action for their support.

Understanding the role of mitochondrial superoxide in biology using a triphenylphosphonium modified manganese (II) SOD enzyme mimic

Geoffrey F. Kelso a, Robin A.J. Smith a and Michael P. Murphy b

Department of Chemistry, University of Otago, P.O. Box 56, 9001,Dunedin, New Zealand b Medical Research Council Dunn Human Nutrtion Unit, Wellcome Trust/MRC Building Hills

Road, CB2 2XY, Cambridge, United Kingdom

In eukaryotic cells mitochondria production is the superoxide radical. We seek to understand the role of mitochondrial superoxide in biological processes by targeting a superoxide dismutase (SOD) enzyme mimic to mitochondria in biological systems. Previous work by our group has shown that the mitochondrial membrane potential (negative inside) drives the uptake of triphenylphosphonium (TPP) modified analogues of vitamin E and coenzyme Q into mitochondria ~'2. A mitochondrially targeted SOD mimic (mitoSOD) has been synthesised by connecting the manganese (II) SOD mimic M40403, developed by Riley et al 3, to the TPP group via a 3 carbon methylene chain. Details of the synthesis, characterization, and biological properties of mitoSOD will be presented.

are responsible for the synthesis of ATP. A byproduct of this oxidative energy

1. Smith R.A.J., Porteous C.M., Coulter C.V., Murphy M.P., Eur. J. Biochem., 263, 709-716, 1999 2. Kelso G.F., Porteous C.M., Coulter C.V., Hughes G., Porteous W.K., Ledgerwood E.C., Smith R.A.J., Murphy M.P., J. Biol. Chem., 276, 4588-4596, 2001 3. Salvemini D., Wang Z., Zweier J.L., Samouilov A., Macarthur H., Misko T.P., Currie M.G., Cuzzocrea S., Sikorski J.A., Riley D.P. Science, 286, 304-306, 1999