Journal of Inorganic Biochemistry 86 (2001) 441

Copper-oxygen complexes for biomimetic oxidation catalysis

V e r a S . I . S p r a k e l a, M a r t i n C. Fe i te rs" , R o e l a n d J .M. N o l t e a, K e n n e t h D. K a r l i n b

" Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands h Department of Chemistry, Johns Hopkins University, Remsen Hall 3400 North Charles Street, MD 21218, Baltimore, USA (e-ma il: vsprakel@sci, kun. n o

The studies described in this poster are aimed at the development of supramolecular oxidation catalysts, in particular models of copper containing natural oxidases, e.g. tyrosinase. To achieve this, mimics 1 and 2a have been designed by combining a binding site for 1,3-dihydroxybenzene guests with a catalytically active dinuclear Cu site, which is capable of dioxygen binding. The Cu(I) ions are each held by pyridyl ligands (TMPA for 1 and PY2 for 2a). The synthesis and characterization of these oxygenase mimics by NMR, UV-Vis and EXAFS techniques are reported along with substrate oxidation studies. In addition, we are aiming at catalysis in a biphasic organic/fluorous phase system for easy catalyst separation as well as enhanced solubility of ~ N ~ . d~. ] 2 c,o4 _ . ~ . ~ [ 2 c,og R: O,. Functionalization ,_._ON-- .e. u c~: ..... -~ of catalyst 2b with k.~(~].~i~ O x ~ r ~ ; ; ~ . > % ~ , ~ _ . _ ~ x x

- - $ i - - fluorinated tails in ~ < N /'-~ O '~'O-'Jff"~-- O . . . . "---" O " ~ ord o isso,vei,, the fluorous phase is in - _ . 7 , ~ - progress, as well as 23 ,z,3 studies on the new ~ " ~ . ~ ' / ' ~ host-guest chemistry in 1 2b:2a: X=Hx=oR . . . . = C6F13 or C8F17

the fluorous phase, x x Acknowledgement: We thank the Dutch Research Council (NWO) for funding through its chemical branch (CW).

× n

S i

Cytochrome bs62 variants: a l ibrary for examining redox potential evolution

Stacy L. Springs, a Susanna E. Bass, a George L. McLendon a Department of Chemistry, Princeton University, Washington Road, Princeton, N J, USA (e-mail. ssprings@princeton.edu)

An understanding of how cytochromes evolve within a given structural archetype to optimize redox potential does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential that occurs through random mutation in the region near the heme of cytochrome b562 ( E . coli). In the first generation of this library, random mutation of F61 and F65, and subsequent examination of a statistically significant sample of the library, demonstrates that the redox potential can vary over 105 mV through mutation at these two positions. The redox potential of the wild type protein occurs at the high potential extremum of the distribution, indicating that F61 and F65 optimally stabilize the reduced state of the protein. At the other extreme, a compositionally conservative set of mutations (F61I/F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. The extrema of the first generation were used as parents of a second generation of variants where random mutations were introduced in place of R98 and R106. This library has a range of redox potential which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands. Again, the redox potential of WT b562 is found at the high potential extremum. The 2.2 A and 2.7 A crystal structures of F61I/F65Y and F61I/F65Y/R106L (low potential variant of the second generation), respectively, will be presented with a corresponding redox-structure analysis. The F61I/F65Y structure indicates that charge-dipole effects introduced by the F65Y mutation and two new internal water molecules are likely responsible for the stabilization of the oxidized protein. Comparisons of experimental and calculated redox potentials for these structures will be presented.

I. Springs S.L., Bass, S.E. and McLendon, G.L.J. Am. Chem. Soc., 39, 6075-6082 (2000).

We gratefully acknowledge Dr. Greg Bowman, Dr. Ilana Nodelman and Prof. C. E. Schutt for assistance with the collection of X-ray crystallographic data and the NIH for financial support.

442 Journal of Inorganic Biochemistry 86 (2001)

T h e r m o d y n a m i c and reactivity studies of interconvertible/~-r/2: r/2 peroxo and bis / l -oxo dicopper species

T. Daniel P. Stack a , Viswanath Mahadevan a, Mark J. Henson a, Jennifer L. DuBois a, Britt Hedman a'b Keith O. Hodgson a'b, Edward I. Solomon a, ~Department of Chemistry, Stanford University, Stanford, California 94305, USA 6Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94309, USA

Simple Cu(I) complexes with predominant nitrogen ligation react with 02 to form many structurally distinct complexes. A particularly intriguing recent example are those systems that exhibit an equilibrium mixture of isoelectronic ~u-rf.'r/Cperoxodicopper(II) (P) and bis /l-oxodicopper(III) (O) isomers. ~2 Such systems provide the possibility of accessing the relative reactivity of these two isomers with externally added substrates. Several peralkylated diamine ligands will be presented that generate equilibrium mixtures of these isomers at -80 ° C in aprotic solvents. Systematic variation of the alkyl-substituents and diamme backbone of the ligands suggests that the equilibrium position is principally controlled by the ligand structure, and that the Cu202N4 core of the bis ,u-oxo isomer is intrinsically more stable than the ¢t-r/e.'r/Cperoxo isomer. Solvents and counteranions play a secondary role in controlling the equilibrium position and the rate of isomer interconversion. Trends indicate that solvent and anion effects are coupled; apolar solvents that favor tight anion association with the dicationic copper complexes preferentially stabilize P, while polar solvents favor O. In one case, the solvent 2-MeTHF causes the rate of isomer interconversion between P and O to be slow relative to rates of formation, decay and reaction with substrates. Kinetic studies in 2-MeTHF reveal that P can not be the progenitor of O, but that an unknown common intermediate must exist. An alternate pathway ofinterconversion between P and O may occur through this intermediate rather than through the generally accepted mechanism of direct reversible cleavage of the O-O bondJ In several reactions examined with suhstrates, P is found to the more reactive isomer at parity of ligand consistent with a more exposed copper/oxygen core. 1..Tolman, W. B. Acc. Chem. Res. 1997, 30, 227-237. 2..Mahadevan, V.; Henson, M. J.; Solomon, E. I.; Stack, T. D. P. J. Am. Chem. Soc. 2000, 122, 10249-10250.

Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563-2606.

Character izat ion of the paramagnet ic intermediates of [NiFe] hydrogenase by means of relativistic D F T calculat ions

Matthias Stein, Wolfgang Lubitz TU Berlin, Max-Volmer-Laboratory of Biophysical Chemistry, Institute of Chemistry, Strasse des 17. Juni 135, D-10623 Berlin, German (e-mail:Matthias.Stein@tu-berlin.de)

Hydrogenases are metalloenzymes which catalyze the reversible heterolytic dissociation of molecular hydrogen. The active centre consists of a heterobimetallic Ni-Fe cluster coordinated by four cysteine amino acid residues, a bridging ligand and three small inorganic molecules at the iron. In several redox states the Nickel atom is paramagnetic and can thus be studied by EPR and ENDOR and related techniques yielding g- and hyperf'me trensors of the magnetic nuclei. These quantities were calculated using relativistic DFT calculations using the ZORA (zero-order regular approximation) and were found to be in good agreement with experimental findings for all paramagnetic intermediates in the reaction cycle of the enzyme [ 1 ]. Aided by these theoretical calculations, a structure-to-function correlation is established [2]. The oxidized, inactive forms Ni-A and Ni-B differ in the extent of protonation of the bridging (oxygen) ligand reflecting their different activation kinetics. In the reduced active Ni-C form, a key intermediate of the reaction cycle, a hydride anion replaces the bridging ligand in the active centre. The light-induced Ni-L state originates from Ni-C by photodissociation of the bridging ligand. The CO-inhibited form binds CO in an axial position at the Ni atom. On the basis of the intermediates found in this way, a plausible reaction mechanism is suggested. Furthermore, an explicit participation of the protein environment in the mechanism is discussed.

1..Stein M., van Lenthe E., Baerends E.J. and Lubitz, W., J. Am. Chem. Soc., in press (2001). 2..Stein, M. and Lubitz, W.; PCCP, in press (2001).

Supported by DFG (grants Lu 315/13 and Sfb 498 TP C2)

Journal of Inorganic Biochemistry 86 (2001) 443

Synthesis, structures and properties of heterobinuclear complexes of a binucleating N6Sz ligand

Gunther Steinfeld,Berthold Kersting Institut fiir Anorganische und Analytische Chemie, Universitiit Freiburg, Albertstr. 21, D-79104 Freiburg, GERMANY (e-mail: steinfel@uni-freiburg, de)

The active site of [NiFe]-hydrogenase from D. gigas consists of a heterobimetallic NiFe complex. While the nickel ion is coordinated by four cysteine sulfur atoms from the protein matrix, the iron is coordinated by two bridging cysteine sulfur atoms and three terminal CO- and CN-ligands, respectively. It is assumed that the free bridging position between the metal atoms plays an important role during the activation of dihydrogen ~. Recently, we reported on the properties of a triply thiolate bridged NiFe-complex of a tripodal ligand with an N6S3 donor set -'. To generate a free bridging position we have now drawn our attention to binuclear complexes of the N682 donor ligand H2L. The new ligand is able to stabilize homo- as well as heterobinuclear transition metal complexes of the type A. The complexes contain a MI(g-SR)2(g-X)M 2 core structure with a free bridging position. In this communication we present the synthesis, structures and properties of the new complexes.

1. J. C. Fontecilla-Camps, S. W. Ragsdale, Adv. Inorg. Chem, 47, 283-333 (1999). 2. G. Steinfeld, B. Kersting, Chem. Commun., 205-206 (2000)

A

Financial support of this work by the Graduiertenkolleg Unpaired Electrons in Chemistry, Physics and Biology is gratefully acknowledged.

Identification of Coq7 as a membrane-bound di-iron carboxylate protein that catalyzes a hydroxylation in the biosynthesis of ubiquinone

PS.1 Stenmark, Jacob Grtinler, Jonas Mattsson, Pavel J. Sindelar, P~ Nordlund, and Deborah A. Berthold Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius vdg 12, S- 106 91 Stockholm, Sweden (e-mail: stenmark@dbb.su.se)

Ubiquinone (UQ) is an essential electron carrier in the respiratory chain of all eukaryotes, and it is also necessary for a functional respiratory chain in prokaryotes. In yeast, mutation of the COQ7 gene results in the absence of UQ biosynthesis and demonstrates a role for this gene in the step leading to the hydroxylation of 5-demethoxyubiquinone (DMQ). Intriguingly, the disruption of the corresponding gene in Caenorhabditis elegans, clk-l, results in a prolonged lifespan and a slowing of development. Due to the pleiotropic effect of this disruption, the small size of the protein, and the iack of obvious homology to other known hydroxylases, it has been suggested that Coq7 may be a regulatory or structural component in UQ biosynthesis, rather than acting as the hydroxylase per se. Here we identify Coq7 as belonging to a family of di-iron containing oxidases/hydroxylases based on a conserved sequence motif for the iron ligands, supporting a direct function of Coq7 as a hydroxylase. We have cloned COQ7 from Pseudomonas aeruginosa and Thiobacillus ferrooxidans and show that when expressed in Escherichia coli, indeed Coq7 complements an E. coli mutant disrupted in an unrelated DMQ hydroxylase gene. Based on the similarities to other well-studied di-iron carboxylate proteins, we propose a structural model for Coq7 as an interfacial integral membrane protein (see figure), and discuss substrate binding and catalysis.

444 Journal of lnorganic Biochemistry 86 (2001)

Influence of nearest neighbors on the conformational equil ibria in DNA bearing a cis-Pt(NH3)2(dGpG) > adduct studied by molecular dynamics s imulations

St6phane Teletch6a, Miguel-Angel Elizondo-Riojas, Gildas Bertho, and Ji~:i Kozelka Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universit~ RenO Descartes, UMR 8601 CNRS, 45 rue des Saints-POres, 75006, Paris 06, France, (e-mail: steletch@biomedicale.univ-paris5.fr)

Molecular dynamics simulations of a DNA duplex crosslinked at a GG sequence by the antitumor drug cisplatin (cis- [PtC12(NH3)2]) have disclosed a possible influence of the bases adjacent to the crosslink on the conformational equilibria.1

The present work investigates the impact of guanines adjacent to the 1,2-GG-Pt crosslink on these equilibria. A

decanucleotide bearing a GGG sequence was treated with the aquated cisplatin form cis-[Pt(NH3)2(HzO)2] 2+, and the two

1,2-GG adducts purified by reverse-phase HPLC. Molecular dynamics simulations of one of the adducts were

accomplished with explicit representation of the solvent. In parallel, an NMR analysis is being carried out in support of

the modeling work.

Elizondo-Riojas and M.-A.; Kozelka, J. submitted.

This work is being supported by the Association for International Cancer Research (grant Nr. 00-321). We are indebted to Johnson-Matthey, Inc., for a generous loan of cisplatin. Computer time from the IDRIS computer center of the CNRS and financial support from COST (project D20/0002/01), enabling scientific exchange with other research groups, are gratefully acknowledged.

Bis[2-benzoylpyrrolato(N-,O)]copper(II): X-Ray structure, solution properties and intercalating ability towards DNA

Giancarlo S t o c c o d , Salvatore Petruso a, Roberta Pierattelli b, Giuseppe Bruno c, Anna Maria Giuliani a, G. Gull.

Department of Pharmaceutical Chemistry, University of Palermo, ViaArchirafi, 32, 90123 Palermo,ITALY Magnetic Resonance Center, University of Florence, Via L.Sacconi, 6,50019 Sesto Fiorentino, ITALY

~Department of Molecular Structure, University of Messina, Salita Sperone,31,98166 Vill. S. Agata, Messina, ITALY

~Department of Inorganic Chemistry, University of Palermo, Viale delleScienze, Parco d'Orleans, 90128 Palermo, ITALY (e-mail:stoccogc@unipa.i 0

The Crystal and Molecular Structures of the bis-chelate Copper(II) complex with 2-Benzoylpyrrole are reported. The Copper(II) ion ,the pyrrole rings and carbonyl groups are coplanar,both coordinating moieties being trans, while the phenyl groups are twisted out of this plane in a propeller- -~ -~ : like fashion.The crystal consists of layers of complex molecules which are interconnected ':<v J ' - - ' ~ : - ~,,~;~ by H-bonds between a C-H of the phenyl group and a coordinated carbonyl of the . . . . " adjacent moiety; in an overall view the crystal appears as a supramolecular array of .~'--:~i mononuclear entities. The structure is reminiscent of the basic crystal architecture shown ? - ~ by copper(II) complexes with 2,2'-bipyrimidine~.The planar Cu-pyrrole portions of the _ ~" ~. "-~ - . . . . . molecules appear to be partially stacked,with a 3,3 A distance between superimposed " . ~ - ' - ' ~ ' ~ ' ~ ' - ~ : ' ~ : " • ' planes,although a shift in the horizontal direction is observed,hence the Cu-Cu distance - ~ _ -,,~ _~'~-~:~ .~ , -.-',,, appears to be 5,132 A.The solution structure of the paramagnetic complex in DMF has """ ~-'~" ~ , - ¢ ~ .... ~,.-'- ~ been examined by NOESY.The intercalating ability of the complex towards DNA has "" been tested by CD.

1. Julve M.,Verdaguer M.,De Munno G.,Real J.A.and Bruno G., Inorg.Chem.,32, 795-802 (1993)

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

Journal of Inorganic Biochemistry 86 (2001) 445

Circular dichroism and magnetic circular dichroism studies of the diferrous form of the R2 subunit of ribonucleotide reductase from mouse

Kari R. Strand a, Yi-Shan Yang b, K. Kristoffer Andersson a & Edward I. Solomon b. aDepartement of Biochemistry, University of Oslo, PB 1041, 0316 Oslo, Norway (e-mail: k.r. strand@biokjemi, uio. no). bDepartement of Chemistry, Stanford University, CA 94305, USA.

The reduction of ribonucleotides to deoxyribonucleotides in living cells is catalyzed by ribonucleotide reductase (RNR). RNR from mouse and E. coli belong to class Ia RNR. Both types of RNR consist of two non-identical subunits, R1 and R2. The active form of their R2 subunit contains a tyrosyl radical and a non-heme iron-oxygen center (Fern-Fern). It is the FeH-Fe H form of R2 that reacts with dioxygen to form the tyrosyl radical essential for catalysis. E. coli R2 has served as a model for RNR in higher organisms, even though a number of significant differences have been observed. The amino acid sequence identity between the E. coli and the mouse enzyme is only about 25%. The iron center in mouse R2 is less stable than that in E. coli, and it can form a stable FeH-Fe m center ~. In this study, binding ofFe H to the binuclear non-heine iron center has been investigated using a combination of circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in the near-IR (NIR) region to elucidate the coordination number and geometry of the FeH-Fe H active site. The dimer ground state magnetic properties is determined via the analysis of the variable-temperature variable-field (VTVH) MCD data. Together these studies on mouse R2 show important spectral similarities and differences to those of other di-iron-oxygen proteins such as the R2 from E. coli and substrate bound (stearoyl-ACP) A9-desaturase 2.

1. Andersson, K. K. & Gr~islund, Adv. Inorg. Chem., 43,359-408 (1995) 2. Yang, Y-S. etal., L Am. Chem. Soc., 121, 2770-2783 (1999)

Support by the Norwegian Research Council, the Iron-oxygen Protein Network (ERBMRFXCT980207) of EU TMR programme and the NSF-Biophysics Program Grant MCD 9816051.

Density functional theory and molecular dynamics results for copper proteins

M. Swart a, M. van den Bosch b, H.J.C. Berendsen c, G.W. Canters b, J.G. Snijders a " Theoretische Chemie (MSC), Rijksuniversiteit Groningen. Nijenborgh 4, 9747 AG, Groningen,

The Netherlands (e-mail: m.swart@chem.rug, nl) b Metalloproteins (Gorleaus Lab), Leiden University, Einsteinweg 55, 2333 CC, Leiden,

The Netherlands c Biofysische Chemie (GBB), Rijksuniversiteit Groningen, Nijenborgh 4, 9747 AG, Groningen,

The Netherlands

The copper protein Azurin (see figure) has been studied by two lines of investigation. One involves the geometry optimization of the protein through a QM/MM approach, where the active site has been treated by Density Functional Theory (DFT) and the rest of the protein by a classical force field. The classical and quantum systems are coupled by a modification of the Integrated Molecular Orbital and Molecular Mechanics (IMOMM) scheme, where the link model has been replaced by the AddRemove model. In this new model, the impact of the artificial link atom has been diminished by removing its interactions: the QM interactions of the link atom are corrected for by MM interactions. Results are given for a number of Azurin molecules: wildtype, mutated and metal substituted. The second line of investigation involves Molecular Dynamics simulations of solvated Azurin, where the force field parameters for the active site have been obtained from DFT

M a IZl

c ~ i s

active site Azurin

calculations on the active sites. These parameters consist of atomic charges (from the Multipole Derived Charge analysis) and force constants (from the IntraFF method). This study shows stable copper sites for both the reduced and oxidized states, and vibrational frequencies that are in the right frequency range (200-450 cm-~). Furthermore, the redox potential for wildtype and mutated Azurin has been obtained from free energy derivatives.

Acknowledgment: NWO/CW, NWO, Unilever Research Vlaardingen.

446 Journal of Inorganic Biochemistry 86 (2001)

Imidazolato Bridged Cuz compounds as model for superoxide dismutase enzyme [Cu(dien)h(X) where X = Im(1), 2-MeIm-(2), Bzlm(3)

P.S. Subramanian a, Winfried Plass b, D.Srinivas c. Central salt and Marine Chemicals Research Institute, Bhavnagar 364 O02,lndia,

(e-mail." sival 4@usa.neO bUnivers i ty o f B i e l e f e l d , F a k a u l t a e t fur Chemie , B ie le fe ld , G e r m a n y c National Chemical Laboratory, Catalysis Division, Pune India

Discrete imidazolato bridged water soluble, especially binuclear Cu complexes are of interest due to the enzyme bovine erythrocyte superoxide dismutase (ZnzCu2BESOD) that catalyses the dismutation reaction of toxic superoxide anion (O,-),.has a histidine bridged Cu(II)-Zn(II) active site. A series of dinuclear [Cu(dien)] complexes with imidazolato derivatives such as imidazolato,(1) 2-Methylinaidazolato (2), ~ ~ ~ Benzimidazolato (3) bridged complexes were synthesised and characterized using various . ' i ~ ! ~ ~ ~ spectroscopical techniques and their structure had been solved by X-ray Diffractometer. ~ ' ~ ' ~' , The Cu-Cu intrametallic distance found varied in the order BzIm (5.82 A) < Im (5.84 A) < 2-Melm (6.0 A) and the corresponding 2J value follows the reverse order Bzlm (38 cm- , ..-' , t)> Im (64cm l ) > 2-MeIm (75cm-t). The contradictory results were indicated that the , existence of significant contribution of intermolecular and anisotropic exchange to 2J values. The stability of the imidazolato bridge was monitored by varying the pH, through Electron Paramagnetic Resonance, UV-Vis spectral techniques from acidic to basic pH and the stability of the bridge found stable between 7.5 to 11. Superoxide Dismutafion activity was determined using NBT Assay method and the relative activities of these compound 1 and 3 was determined as 1.5% and 0.3% as that of the BESOD. References: 2. Hideki Ohtsu, Yuichi Shimazaki, Akira Odani, Osamau Yamauchi, Wasuke Mori, Shinobu Itoh and Shunichi

Fukuzumi, J.Am.Chem. Soc, 122, 5733-5744, (2000)

Complexation of antimony(Ill) and trypanothione: formation of a novel tertiary complex

Hongzhe Sun, Siucheong Yan, and Keyang Ding Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China (e-mail: hsun@hku.hk)

Leishmaniasis is affecting more than 10 million people worldwide. Several antimony compounds have been used for the treatment of leishmaniasis for many decades. However the mechanisms of action of the antimony drugs is still remains unknown. One of the major target of the antimony drugs is thought to be trypanothione, which is a major low molecular mass thiol inside the parasite, and overproduction of trypanothione in cells is related to resistance of antimony drugs. ILJ

~1 IIl [2] Il l Here we report the first chemical characterization of Sb -trypanothione complex. Sb bounds to trypanothione at two thiolate of the Cys residues and also possibly a water, the binding is pH dependent and is strongest at biological pH ( ca. 7 ) with the stability constant log K 23.57 at 298 K. Addition of small monothiol ligands such as glutathione and cysteine to the Sbm-T(S2) complex leads to the formation of a novel tertiary complex as judged from NMR & ESI-MS data. Thiolates from both trypanothione and monothiol simultaneously bound to Sb |I1 center. The discovery of the tertiary complex is important as it may play a role in the transport of antimony and the antileishmaniasis properties of the drugs are probably via the formation of trypanothione reductase with Sbm-trypanothione. We acknowledge the financial support from the University ofHong Kong.

NH~ ., O .

SH >-t H N.H~

o ~ H o / - O O ~ N ~ N V ~ N ?

NH a H O H

Trypanothione, T(SH)2

1. Mukhopadhyay, R., Dey, S., Xu, N., Gage, D., Lightbody, J., Ouellette, M., Rosen, B. P., Proc. Natl. Acad Sci. USA, 1996, 93,10383-10387.

2. Yan, S., Ding, K., Zhang, L., Sun, H., Angew. Chem. Int. Ed, 2000, 39, 4260-4262.

Journal of Inorganic Biochemistry 86 (2001) 447

Supramolecular catalysts for light-induced production of fuel, based on the principles of natural photosynthesis

Licheng Sun a, Bj6m/~d~:ermark a, Leif Hammarstr6m b, Stenbj6m Styring c Department of Organic Chemistry, Arrheniuslab, Stockholm University, SWEDEN

(e-mail: licheng.sun@organ.su.se) b Department of Physical Chemistry, Uppsala University, Box 532, 751 21 Uppsala, SWEDEN c Department of Biochemistry, Center for Chemistry and Chemical Engineering, University of Lund, Box 124, S-221 O0 Lund, SWEDEN)

In the photosynthesis of the green plants, light is used to transfer electrons from water, which is oxidized to molecular oxygen, to carbon dioxide, which is reduced to biomass. One crucial part of the photosynthetic apparatus is Photosystem iI (PS II), a membrane bound enzyme, where electrons are released from excited chlorophyll, transferred to Photosystem I and ultimately utilized in carbon dioxide reduction. In this process, chlorophyll is oxdized, then regenerated by electron transfer from water via a tyrosine unit and a cluster of four manganese ions. The goal of our work, which will be presented, is to synthesize and study artificial mimics of PSII, which use tris(bipyridine) ruthenium complexes as photosensitizers in place of chlorophyll. So far, we have been able to syntesize a supermolecule, in which a dinuclear manganese cluster is linked to a ruthenium sensitizer via a tyrosine molecule. On irradiation with visible light in the presence of an external acceptor, this complex is capable of transfering three electrons , at least, from the manganese cluster to the acceptor via the ruthenium complex. In the process, the manganese cluster is oxidized from Mn(II)(II) to Mn(III)(IV), to be compared with the four electron process of PSII. To increase the valence of Mn, phenolate groups are introduced in the ligands. Because high valent manganese complexes are strong oxidants, we have also studied epoxidation and other oxidation reactions, using our manganese complexes as catalysts. 1. Magnuson, et al. J. Am. Chem. Soc., 121, 89-96(1999). 2. Sun, et al. J. Am. Chem. Soc., 121, 6834-6842(1999). 3. Sj6din, et a]. J. Am. Chem. Soc., 122, 3932-3936(2000). 4. Sun, et al. Chem. Soc. Rev., 30, 36-49(2001).

Dioxygen reactivity of copper(I) complexes with a tridentate ligand having 6-methylpyridyl groups

Masatatsu Suzuki. Yuji Tomii, Masaichi Terada, Hideki Hayashi, Hideki Furutachi, Shigenori Nagatomo, a Shuhei Fujinami, Seiji Ogo, a Yoshihito Watanabe, a and Teizo Kitagawa a Department of Chemistry, Kanazawa University, Kanazawa, 920-1192, Japan (e-mail. suzuki@cacheibm.s.kanazawa-u.ac.jp 0

a Institute of Molecular Science, Okazaki, 444-0863, Japan

Previously we found that introduction of 6-methyl group(s) into pyridyl group(s) in tpa has a significant influence on the dioxygen reactivity of the copper(l) complexes, l In this study we investigated the reactivity of a copper(I) complex with a tridentate ligand containing 6-methylpyridyl groups with dioxygen, [Cu(Ll)] + (1) (Lt=bis(6-methyl-2- pyridylmethyl)benzylamine). 1 reacted with dioxygen in dichloromethane at -78°C to produce both ktS_ r/Z;r/Z-peroxo dicopper(lI) and bis(~u-oxo)dicopper(III) species, which were identified by UV-vis and resonance Raman spectroscopies. Thermal decomposition of 1 in dichloromethane resulted in the oxidation of L1; hydroxylation of 6-methyl group, oxidation of 6-methyl group to aldehyde, coupling of two L 1 at 6-methyl position, and N-dealkylation (6- methylpyridylmethyl group). Decomposition of 1 containing deuterated ligand (L1, Li-d6, or Ll-d 10) showed KIE (D/H) o f - 3 at -20°C, suggesting that the oxidation proceeds via hydrogen-abstraction of the methyl or methylene group of 6- methyl-2-pyridylmethyl pendant. Details of the thermal decomposition study will be presented.

1. Hayashi, H.; Fujinami, S.; Nagatomo, S.; Ogo, S.; Suzuki, M.; Uehara, A.; Watanabe, Y.; Kitagawa, T. J. Am. Chem. Soc. 2000, 122, 2124.

448 Journal of Inorganic Biochemistry 86 (2001)

Electron transfer and nitrite reduction processes of multicopper nitrite reductase

Shinnichiro Suzuki a, Kunishige Kataoka b, and Kazuya Yamaguchi a "Department o f Chemistry, Graduate School o f Science, Osaka University, Toyonaka, Osaka 560-0043, JAPAN ( e-mail:bic@ch.wani, osaka-u.ac.jp) bDepartment of Chemistry, Faculty of Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, JAPAN

Dissimilatory nitrite reductase (NIR) is a key enzyme in the anaerobic respiratory pathway of denitrifying bacteria, catalyzing one electron reduction of nitrite to NO. Cu-containing NIR is a trimer with one type 1 Cu (blue copper) bound in one subunit and one type 2 Cu (nonblue copper) located between the adjacent subunits) The type 1 Cu site bound by 2His, Cys, and Met accepts one electron from an electron donor protein and transfers it to the reaction center, type 2 Cu having 3His and a solvent ligands. The intramolecular electron-transfer process from type 1 Cu to type 2 Cu would be closely linked to the following catalytic reduction process of the substrate. The Asp and His residues around the type 2 Cu site not only control the electron-transfer process but also provide protons for nitrite bound to type 2 Cu as a general acid- base catalyst in the nitrite reduction process. 2

Moreover, we have recently found a novel NIR, which has an extra type 1 Cu with the usual set of the type 1 Cu and type 2 Cu sites. The cloning, expression, and mutagenesis of the enzymes was carried out. The visible absorption spectrum of the native enzyme is composed of those of the two type 1 Cu sites.

1. Suzuki S., Kataoka K., Yamaguchi K., Inoue T., and Kai Y., Coord. Chem. Rev., 190-192,245-265 (1999). 2. Suzuki S., Kataoka K., and Yamaguchi K., Acc. Chem. Res., 33,728-735 (2000).

The Ministry of Education, Science, Sports and Culture of Japan is acknowledged for financial support.

Effects of acylphosphatase on the ion transport mechanism of the Na+,K+-ATPase

Francesco Tadini Buoninsegni a, Andrea Dolfi a, Maria Rosa Moncelli a, Chiara Nediani b, Paolo Nassi b, Rolando Guidelli a

Department of Chemistry, Polo Scientifico-University of Florence, Via della Lastruccia 5, 50019 Sesto Fiorentino, ITALY (e-mail." francesco@gda l.chim, unifi.i 0 b Department of Biochemistry, University of Florence, Viale Morgagni 50, 50134 Florence, ITALY

The aim of the present study is to investigate whether acylphosphatase, a widespread cytosolic enzyme of about 1 lkDa, may affect the functional properties of the shark Na+,K+-ATPase in terms of ion transport. This research is carried out using a novel experimental method, which has been recently developed to perform concentration jumps of an arbitrary

~2 substrate at the surface of a solid supported membrane (SSM) ' . The SSM consists of an alkanethiol monolayer firmly anchored to the gold surface via the sulphydryl group, with a second phospholipid monolayer on top of it. Membrane fragments containing shark Na+,K+-ATPase are adsorbed on the SSM. Upon adsorption, the ion pumps are activated by performing concentration jumps of ATP at the surface of the SSM, and the electrical currents generated by the Na+,K +- ATPase are measured under short-circuit conditions. To investigate the effect of acylphosphatase on the ion transport by the Na+,K+-ATPase, the SSM technique is employed to carry out ATP concentration jumps in the presence and in the absence of acylphosphatase, and acylphosphatase concentration jumps in presence of ATP and Na ÷ ions. The reported results clearly indicate that acylphosphatase can induce significant modifications in the transport mechanism of the Na',K+-ATPase. Possible mechanisms for such an effect are discussed.

1. Pintshovius J. and Fendler K., Biophys. J., 76, 814-826 (1999) 2. Pintshovius J., Fendler K. and Bamberg E., Biophys. J., 76, 827-836 (1999)

Journal of Inorganic Biochemistry 86 (2001) 449

Adduct formation by platinum(II) complexes of ligands containing aromatic rings

M a s a k o Takani a, Ta t suo Y a j i m a b, Ak i ra Odani c, O s a m u Y a m a u c h i d ~Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machL Kanazawa 920-1192, JAPAN (e-mail: takani@dbs.p.kanazawa-u.acjp) bGraduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, JAPAN CResearch Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, JAPAN dUnit of Chemistry, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, JAPAN

Aromatic ring stacking and hydrogen bonding are important factors for selective formation of mixed ligand complexes and of adducts with uncoordinated molecules with relevant interacting groups. In order to clarify the structural dependence of adduct formation between planar complexes with aromatic rings and aromatic molecules with charged or polar groups, we studied syntheses and structures of Pt(II) complexes containing an aromatic diimine (DA) and an ethylenediamine derivative with various side chains (ben), Pt(DA)(ben) (DA = 2,2'-bipyrimidine (bpm), 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen); ben = N,N'-dibenzylethylenediamine (dben), N,N'-bis(4-hydroxybenzyl)- ethylenediamine (hben)), and adduct formations with uncoordinated molecules (L - phthalate (PA), gentisate (GA), etc.). The Pt(II) complexes formed such adducts with L as [Pt(phen)(dben)](GA)2 and [Pt(bpm)(L-Arg)](L) (L = PA, terephthalate (TA), etc.; Arg = arginine). ~H NMR spectra exhibited small upfield shifts of the protons of GA molecules, one of which was disclosed to partly stack with coordinated phen in the solid state. The carboxylate moiety of the stacked GA molecule in [[Pt(phen)(dben)](GA)2 was found to be hydrogen bonded to the amino group of dben of a neighboring complex unit, while the benzyl rings were directed away from the Pt(II) center and free from any meaningful interactions. A similar hydrogen bond was also observed for [Pt(bpy)(dben)](GA)2, which may be of significance for the binding of Pt(II) and other metal complexes of amine ligands to proteins and DNA. No intramolecular stacking was observed for [Pt(dben)2]C12. These results indicate that planar Pt(II) complexes with aromatic tings may form adducts with various small molecules and serve as receptors in intermolecular interactions.

Reactivity of bis(/.t-oxo)dicopper(III) complex toward external substrates

M a s a y a s u Taki b, Y o s h i m i t s u Tachi a, Shunichi Fukuzumi b, Shinobu I toh a

Department of Chemistry, Graduate School of Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan (shinobu@sci.osaka-cu.ac.jp)

b Department of Material and Life Science, Graduate School o f Engineering, Osaka University, CREST, Japan Science and Technology Corporation, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan (e-mail: taki@chem.eng, osaka-u.ac.jp )

Reactivity of a bis(~t-oxo)dicopper(III) complex supported by bidentate ligand L (L = N-ethyl-N-[2-(2- pyridyl)ethyl]-ct,ct-dideuteriobenzylamine) has been investigated to provide insight into the reactivity 9~J~ ~J of dinuclear non-heine transiton metal-oxo species. Hydorogen atom abstraction from 1,4- o T " cyclohexadiene and 10-methyl-9,10-dthydroacridine by the bis(,u-oxo)dicopper(III) complex proceeds . , ~ f u efficiently to give be~tzene and lO-methylacridinium ion, respectively. The reaction rate is second- "~ order with respect to the concentration of bis(/3-oxo)dicopper(III) complex and first-order on the substrate concentration. Such an unusual kinetic behavior observed in the present reactions implicates L existence of a new copper-active oxygen intermediate that is responsible for the C-H bond activation of the external substrates.t Oxygenation of sulfides to the corresponding sulfoxide by the bis(/~-oxo)dicopper(III) complex also proceeds quantitatively. Detailed kinetic analysis has revealed that the oxidation proceeds via a direct oxygen atom transfer mechanism via formation of binary complex between the substrate and the metal-oxo species. Mechanistic details of each reaction will be discussed based on the kinetic data.

1. Taki, M.; Itoh, S.; Fukuzumi, S. J. Am. Chem. Soc., 123, in press (2001)

450 Journal of lnorganic Biochemistry 86 (2001)

The metal ion cofactor of bacillus subtilis oxalate decarboxylase is Mn(II)

Adam Tanner. Stephen Bornemann Biological Chemistry Department, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH. (e-mail." adam.tanner@bbsrc.ae.uk)

Oxalate decarboxylase (ODC) (EC 4.1.1.2) catalyses the decarboxylation of oxalate to formate and carbon dioxide. Curiously, although oxygen is not consumed or evolved in the reaction it is required for activity, implying the presence of redox active cofactors. We have shown B. Subtilis ODC to be the first example of this enzyme from a bacterium [ I ]. It is a member of the cupin family, which is defmed by the presence of conserved motifs in the enzyme's primary sequence. Since ODC contains a duplication of these motifs in each monomer it is referred to as a bicupin. Three genes coding for bicupins and sharing significant sequence similarity with the first ODC to be isolated (from the wood-rotting fungus Collybia velutipes) were identified in the genome of B. subtilis. Oxalate decarboxylase activity was detected in cells grown at an acidic pH: optimal activity was observed in cultures grown at pH 5 [1]. B. subtilis ODC was purified to homogeneity through a series of ion exchange chromatography steps and gel filtration. The N-terminal sequence of the pure enzyme was determined and it revealed the gene yvrK to be responsible for ODC production. SDS-PAGE indicated the subunit mass to be approximately 44 kDa, consistent with the prediction from the primary sequence of YvrK. The mass of the native enzyme by gel filtration was estimated to be 254 kDa, suggesting the native enzyme to be a hexamer. The UV/visible spectrum of pure ODC showed normal absorbance for a protein with no additional chromophore. YvrK is the first oxalate-degrading cupin to be successfully cloned and over-expressed in an active form, providing the opportunity to study this enzyme in detail. EPR and XANES spectroscopies and metal analysis have shown B. subtilis ODC to be a Mn(II)-containing enzyme. Results from EPR and EXAFS spectroscopies are consistent with structural predictions of the Mn(II) environment based on homology with Mn(II) oxalate oxidase [2]. The presence of Mn(II) in this enzyme provides an explanation for the role of dioxygen in the reaction: it may be required as a cofactor, acting as an electron sink during catalysis. 1. Tanner, A. and Bornemann, S. (2000). J. Bacteriol. 182, 5271-5273. 2. Requena, L. and Bornemann, S. (1999). Biochem. J. 343, 185-190.

Molecular recognition of proteins. Inhibitory effects on the chiral recognition of a protein for metal complex anions

Toshiaki Taura Laboratory of Molecular Recognition and Coordination Chemistry, Faculty of Information Science and Technology, University of Aichi Prefecture, Nagakute, 480-1 198 Aichi, JAPAN (e-mail: taura@ist, aichi-pu.ac.jp)

How does protein recognize the shape or chirality of small molecules ? The answer to this question should help to clarify the mechanism of molecular recognition in biological systems. Recently, we have reported that bovine serum albumin recognizes the chirality of tris(oxalato)- cobaltate(III) anions in aqueous solution, and that the protein interacts with the A-isomer of the complex anion selectively. 1 It was found on the basis of circular dichroism (CD) data that the interaction could be caused by the electrostatic force. In this work, we investigated the inhibitory effect for this system in order to obtain more detailed information about chiral recognition phenomena of proteins.

1. Taura, T., Inorg. Chim. Acta, 252, 1 - 3 (1996)