Journal of Inorganic Biochemistry 86 (2001) 381

Structural characterisation off-d hetero-bimetalic Ln(III)-Zn(II) cryptates

Carlos Pla tas- Ig les ias , Raque l Rodr iguez-Cor t i f ias , Fernando Aveci l la , Andr r s de Bias, Teresa

Rodr iguez -Blas

~' Departamento de Quimica Fundamental, Facultad de Ciencias, Universidade da Coru~a, Campus da Zapateira sin, 15071 A Coru~a, SPAIN. (e-mail. mayter@udc, es)

As a part of our effort to prepare Ln(III) complexes with interesting photophysical properties, in previous works we reported the synthesis, structural characterization (both in solution and in the solid state) and luminescence properties of mononuclear Ln(III) cryptates with formula [LnL(NO3)](NO3)2. l' 2 The deprotonation of the phenolic oxygen atoms of the ligand (see figure), allows the preparation of f-d heterodinuclear complexes Ln(III)-Zn(II), whose structural characterisation both in the solid state and in aqueous solution will be presented and discussed.

1. Platas C., Avecilla F., de Blas A., Rodriguez-Blas T., Geraldes C. F. G. C., T6th E., Merbach A. E. and BiJnzli J.-C. G., J. Chem. Soc., Dalton Trans., 611 - 618 (2000)

2 Platas C., Avecilla F., de Blas A., Geraldes C. F. G. C., Rodriguez-Blas T., Adams H. and Mahia J., Inorg. Chem., 38, 3190- 3199 (1999)

CH3

I CH:3

Authors thank Universidade da Comfia for financial support.

Modelling the active site environment of vanadium haloperoxidases: Dioxovanadium(V) complexes with functionalized N-salicylidenehydrazides as

tridentate ligands

Axel P o h l m a n n a, Winf r i ed Plass a'b.

Fakultiit ffir Chemie der Universitiit Bielefeld, Postfach 10 O1 31, D-33501 Bielefeld, Germany '~ Current address. Anorganische Chemie, Universitgit Siegen, Adolf-Reichwein-Strafle, D-57068

Siegen, Germany, (axel.pohlmann@uni-bielefeld.de)

The protonation of the vanadate moiety in vanadium containing haloperoxidases is still a matter of debate. All the suggested reaction pathways contain a proton transfer step at the vanadate center. Only a few model complexes contain acid-base acceptor groups supporting both protonated and deprotonated forms of relevant cis-dioxovanadium(V) com- plexes. Schiff base ligands derived from salicylaldehyde and co- hydroxycarbonicacid hydrazids offer an approach to readily functionalized vanadium(V) complexes, which can easily be deprotonated) The influence of the protein environment can be modeled by the variation of the ligand side chain. The ammonium salts and the corresponding protonated neutral dioxovanadium(V) complexes of these ligands with different side chain length were isolated. In the solid state the ammonium ion takes part in the supramolecular environment of the dioxo- vanadium moiety. The characteristics and reactions of t h e s e compounds will be discussed.

2. Plass W, Pohlmann A. and Yozgatli H.-P, J. Inorg. Biochem, 80, 181 - 183 (2000)

382 Journal of Inorganic Biochemistry 86 (2001)

Spectroscopic studies of an Fe(IIl) intermediate of ACC oxidase

Codrina V. Popescu a Amy M. Rocklin a, Luca Quaroni b, William B. Antholine c, Michael P. Hendfich d, Lawrence Que, Jr. b, , and Eckard Mtinck a and John D. Lipscomb a " Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,

Minneapolis, MN 55455; h Department of Chemistry, University of Minnesota, Minneapolis, MN 55455; CMedical College of Wisconsin, Milwaukee, W1 53226; aDepartment of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213

The final step of ethylene biosynthesis, the oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, HCN, and CO2 is catalyzed by ACC oxidase (ACCO). In vitro, ACCO requires the addition of exogenous Fe(II), along with 02, CO2, and ascorbate. J Aerobic incubation of Fe(II)ACCO with ACC results in the formation of 0.35/0.05 mol C2H4/mol Fe(II)ACCO. Under these conditions Fe(II)ACCO is rapidly oxidized to Fe(III)ACCO, exhibiting an unusual rhombic EPR signal, with g-values at 4.77, 4.30 and 4.05 (at 9.6 GHz). These values are inconsistent with any isolated S = 5/2 system under the assumption that go is near g = 2. Simulations of the 4.2 K M6ssbauer spectrum of this species suggest that these three resonances belong to the middle doublet of a unique species with -0.7 << D << -0.4 cm 1. If this range is correct, then the higher field resonances in the EPR spectrum might not be accurately calculated with the assumption of D >> g/~BB, but S-band EPR reveals the same g-values. Because the oxidation of the iron may lead to a new species with radical character, we are exploring the possibility of spin interactions in the active site in addition to fourth order zero- field splitting terms. I. Rocklin, A. M., Tierney, D. L., Kofman, V., Bmnhuber, N.M.W., Hoffman, B.M., Christoffersen, R.E., Reich, N.O., Lipscomb, J. D. & Lawrence Que, Jr. (1999) Proe. Natl. Acad. Sci. USA 96, 7905-7909.

This work was supported by NIH Grant GM-24689.

Theoretical study of the mechanism for the catalytic cycle of pea seedling amine oxidase (PSAO)

Rajeev Prabhakar, Per E. M. Siegbahn Department of physics, Stockholm University, Box 6730, S-11385 Stockholm, Sweden (e- mail: raju@physto, s e)

Pea seedling amine oxidase (PSAO) catalyzes the oxidative deamination of primary amines to aldehydes, accompanied by the reduction of molecular oxygen into hydrogenperoxide in two half-reactions known as the reductive and oxidative half-reactions. The entire catalytic mechanism of PSAO has been investigated quantum mechanically using hybrid density functional theory (B3LYP). The reductive half-reaction is suggested to be divided into six steps where the C-H activation of the substrate is rate limiting step in agreement with the experiment. The reductive half-reaction is calculated to be exothermic by 17.4 kcal/mol. The oxidative half-reaction is suggested to occur in eight steps. In the dioxygen activation process the formation of a superoxide ion that leads to the creation of a radical-pair is experimentally suggested to be rate limiting. The present calculations suggest that the creation of the radical-pair induce a spin-flipping by spin-orbit coupling and transform the system from a triplet to an open shell singlet state in the rate determining step. In the rest of the steps a mechanism for the formation of ammonia is suggested and the results are compared to experimental findings.

Journal of Inorganic Biochemistry 86 (2001) 383

Reactivities of Fe(III) and Co(III) model complexes with carboxamido nitrogens and thiolato sulfurs as donors: relevance to nitrile hydratase

Pradip K. Mascharak, Juan C. Noveron, Marilyn M. Olmstead and Laurie A. Tyler Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064,

USA, (e-mail: mascharak@chemistry, ucsc, edu.)

Assimilation of organic nitrile by several microorganisms is facilitated by the enzyme nitrile hydratase. The enzyme contains either a non-heme Fe(III) or a non-corrinoid Co(III) center at the active site. Recent crystallographic studies have revealed that the single iron site in the al3 heterodimer is coordinated to two deprotonated carboxamido nitrogens and three Cys-S centers and two of the three bound ~ • ° Cys-S centers are post-translationally modified to Cys-sulfenic and -sulfinic groups. The ~ _ ~ _ ~ L ~ o " ~ [ / ~ / ' ~ structure of the Co(III) site is presumed to be similar to that of the iron site. We have synthesized the Fe(III) and Co(III) complex of a designed ligand PyPSH4 that contains 1 ~ two carboxamide and two thiolate groups in its framework. The structures, spectroscopic properties and redox properties of these two model complexes have been determined. The reactions of these model complexes with dioxygen have been studied. We have also measured the pK, of a bound water molecule to the Fe(III) site. Binding of various ligands at the M(III) sites has been explored. We have also been able to employ the Co(III) model in promoting catalytic hydrolysis ofnitriles. These results will be reported and discussed in relation to the enzyme.

Spectroscopic studies of the active site ofparacoccus denitrificans nitric oxide reductase

Louise A. Prior, Myles R. Cheesman, Andrew J. Thomson, Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, NR4 7T J, United Kingdom

Nitric oxide reductase (NOR) catalyses the one electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of proteins called heme-copper oxidases (HCOs). The main feature distinguishing NOR from the HCOs is the elemental composition of the active site; the dinuclear centre comprises heine bs interacting with non-heine iron (FEB) rather than CuB.

Low temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopy, have been used to study the enzyme in the oxidised, partially reduced, (~ reduced, and reduced CO bound (2) states. In several oxidation states NOR exhibits novel properties which are not generally observed for HCOs.

The WT form of the enzyme has been studied at both RT and LT confirming the presence of two LS ferric heroes with bis-HIS and HIS-MET ligation, and a HS heme antiferromagnetically coupled to a non-heme iron at the active site. Additionally an unusual form of the enzyme has been discovered, and attempts to characterise this have begun.

1. Gronberg, K. L. C., Roldan, M. D., Prior, L., Butland, G., Cheesman, M. R., Richardson, D. J., Spiro, S., Thomson, A. J. and Watmough, N. J. (1999) Biochemistry 38, 13780-13786 2. Hendriks, J. H. M., Prior, L., Baker, A. R., Thomson, A. J., Saraste, M. and Watmough, N. J. (2001, Biochemistry submitted)

The EPSRC is acknowledged for financial support.

384 Journal of Inorganic Biochemistry 86 (2001)

Redox centers involved in oxygen chemistry catalyzed by cytochrome oxidase.

Denis A. P r o s h l y a k o v a, Jose Cerda a, Miche l le A. Press ler a, Cather ine D e M a s o a, Joseph F. L e y k a m b, Dav id L. DeWit tb ,Gera ld T B a b c o c k a

Department of Chemistry (email: denisp@cem.msu.edu) and b Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,

Michigan, 48824, USA

We have been characterizing the O-O bond cleavage by cytochrome c oxidase (CcO), which occurs as a one-step reduction process resulting in a ferryloxo compound on heme a 3 at the peroxy oxidation level (Pro). Based on mutagenisis and X-ray crystallography data, we have proposed that the amino acid residue,Tyrz44, is likely to be oxidized in this process I. Such a proposed tyrosine radical is expected to be inaccessible to traditional EPR spectroscopy because of its close proximity to the redox-active CUB. Therefore, we undertook to label the putative radical by chemical means (~2sI) and the results show that Tyr244 is indeed the predominant labeled protein site in Pm 2. We have detected a UV optical absorption signature in Pm, which may be unique to this species. A similar but red-shifted signal is detected in F' species, another radical-containing form of cytochrome oxidase where EPR and labeling evidence suggests that the Tyr radical is replaced with a Trp radical, It is proposed that the two signals are due to absorption by the Tyr and Trp radicals in Pm and F', respectively. We found that O2 reduction CcO is reversible. The FetI-o2 oxy-like species is easily reformed from FeW=O ferryloxo intermediate (Pr of F) by a ns-laser pulse. The O-O stretching mode at 590 cm ~ is higher than that of the normal oxy species, suggesting altered geometry. Similarly, previously unobserved ferric-peroxy species was generated from a ferric hydroxy form of CcO. Both photoreactions occur faster than 108 s -t, involve two-electron oxidation of oxygen and reduction of heine a. Such reaction was not observed for the Pm species. Our findings establish cytochrome oxidase in the family of metallo-radical enzymes and emphasize the importance of proton-controlled transfer of oxidizing equivalents in the protein moiety as a strategy for efficient energy utilization in coupling to proton-pumping events. i. Proshlyakov et al., Proc. Natl. Acad. Sci. U.S.A. 95, 8020-8025 (1998) 2. Proshlyakov et al. Science 24, 1588-1591 (2000)

Development of a paramagnetic probe for the study of protein-protein complexes

Miguel Prudencio a, Jan R o h o v e c b, Joop Peters b, Aldr ik Velders a, Jaap H a a s n o o t a, Mar t in Verbee t a,

Marce l lus U b b i n k a

Gortaeus Laboratories, Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands. b Laboratory for Applied Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands. (e-mail. m.prudencio@chem.leidenuniv, nl)

The study of protein-protein complexes is of great importance for the understanding of a variety ot~ biological processes. During the last decade experiments were developed that allowed NMR techniques to be used in the study of paramagnetic proteins ~. The use of paramagnetic NMR has been extended to the characterisation of protein-protein interactions of highly transient complexes. The method is based on paramagnetic effects caused by a metal atom in one protein of the complex, which change the NMR spectrum of the other protein 2.

Work is being carried out towards the development of a paramagnetic probe that can be specifically attached to one of the proteins in a transient complex. The intermolecular paramagnetic effects thus caused on the other protein can be used to gather structural information about the complex. Putative paramagnetic probes have been synthesised and their ability to bind specific residues on the protein surface is currently being studied.

1. Bertini, I., Rosato, A., Turano, P., Pure Appl. Chem., 71, 1717-1725 (1999) 2. Ubbink, M., Ejdeb/ick, M., Karlsson, B.G., Bendall, D.S., Structure, 6, 323-335 (1998)

This work was supported by the EU Research Training Network (Transient Network), Contract N.- HPRN-CT-1999- 00095

Journal of lnorganic Biochemistry 86 (2001) 385

Interactions of anionic carboranylated porphyrins with DNA.

Rober to Purrel lo a, R os a r i a Laucer i a, Shankar J .Shet ty b and M. Gra~a H.Vicen te b

" Dipartimento di Scienze Chimiche Universith di Catania, Viale Andrea Doria 6 95125 Catania, ITALY (e-mail: rpurrello@dipchi, unict, it)

b Departments of Chemistry and Neurological Surgery, University of California Davis, CA 95616, USA

Porphyrins have been widely investigated as anti-cancer drugs in photodynamic therapy. The formation of porphyria- DNA complexes, which is believed to be an important step leading to anti-tumor activity, ~ _ o.H ~ is known to be facilitated by the electrostatic attractions between the periphery of cationic porphyrins and the anionic phosphate backbone. On the other hand, the interaction of anionic molecules with DNA has not been as well studied. 1 Recently, water-soluble anionic nido-carboranyl-containing porphyrins have been proposed as boron delivery o ~e drugs in Boron Neutron Capture Therapy (BNCT) of tumors. The present work was performed with the aim of investigating whether the negative charges and the bulkiness of the nido-carboranyl groups at the periphery of these molecules influenced the 2 , , ~ - formation of porphyrin-DNA complexes. In this study we have characterized the acid- base equilibria of meso-tetrakis[4-(nido-carboranyl)phenyl]porphyrin and meso-tetrakis[3-(nido-carboranyl)phenyl] porphyrin and investigated their interactions with DNA. We present evidence that these tetra-anionic molecules do indeed interact with DNA under physiological conditions. Furthermore, we show that the counterintuitive binding of these tetra- anionic porphyrins to a 2-fold array of negative charges is driven by molecular recognition processes that can be deeply affected by the spatial distribution of the anionic substituents.

1. Y. Li, R.Geyer, D. Sen, Biochemistry, 35, 6911-6922 (1996); b) S. R. Chatterjee, T. S. Srivastava, J. P. Kamat, T. P. A.. Devasagayam, J. Porphyrins Phthalocyanines, 2, 337-343, (1998).

BisOt-oxo)dimanganese complexes by tripodal ligand design

Daniel Pursche, Michael U. Triller, Nicole Reddig, Florian Schweppe, Annette Rompel, Bemt Krebs. institute of lnorganie Chemistry, University of Miinster, Wilhelm-Klemm-Str. 8, 48149 Miinster,

GERMANY (e-mail." pursche@uni-muenster, de)

Manganese ions in different oxidation states are involved in redox processes in the active sites of many metalloproteins such as manganese catalase or the oxygen evolving complex of photosystem II.1 It is our goal to investigate the variation of biomimetic properties of tripodal ligands that are utilized in the synthesis of model complexes for manganese containing proteins. Methylimidazole is often used to mimic histidine in model compounds 2 but it is of great interest if other heterocyclic aromatic moieties can be regarded as similarly efficient models. ~, We used tripodal ligands containing pyridine, picoline and quinoline to synthesise the . . . . ~5 corresponding bis(p-oxo)dimanganese complexes. The donor sets of these ligands contain one aliphatic and three aromatic nitrogen donor atoms. Although the same reaction conditions ~' were applied, the complexes were obtained in different oxidation states (III/III and III/IV respectively). Bulkier ligands appear to favour the formation of the dimanganese(III,III) complexes, but electronic effects will also influence the oxidation state. The data obtained from our spectroscopic and crystallographic characterization will be used as a basis to predict the oxidation states of such systems with the help of theoretical calculations.

1. Barynin V. V., Hempstead P. D., Vagin A. A., Antonyuk S. V., Melik-Adamyn W. R., Lamzin V. S., Harrison P. M., Artymiuk P. J., J. Inorg. Biochem, 67, 196 (1997).

2. Pascaly M., Duda M., Schweppe F., Zurlinden K., Mtiller F. K., Krebs B., J. Chem. Soc., Dalton Trans., 828-831 (2001)

Financial support by the Deutsche Forschungsgemeinschafl (DFG) is acknowledged.

386 Journal of Inorganic Biochemistry 86 (2001)

S t r u c t u r a l c h a n g e s a s s o c i a t e d w i t h c a l c i u m - d e p e n d e n t a c t i v a t i o n o f t h e d i - h e m e

c y t o c h r o m e c p e r o x i d a s e o f Paracoccus pantotrophus

Yan Qiu, c Sofia R. Pauleta, a'b Yi Lu, ~ Celia F. Goodhew, b Isabel Moura, a Graham W. Pettigrew, b John A. Shelnutt c'd

~ Centro de Quimica Fina e Biotecnologia, Departamento de Quimica, Faculdade de Cidncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal

b Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH

C Department of Chemistry, The University of New Mexico, Albuquerque, NM 87131 J Biomolecular Materials and Interfaces Department, Sandia National Laboratories, Albuquerque,

NM 87185-1349

The structural changes in the macrocycle, substituents, and axial ligands of the heme caused by Ca 2+ binding to the diheme cytochrome c peroxidase from Paracoccus pantotrophus are rev ea l ed by resonance Raman spectroscopy of the oxidized, mixed valence, and reduced forms of the enzyme. For the inactive oxidized form, ~ the changes in the macrocycle vibrational modes indicate a Ca2+-dependent increase in the out-of-plane distortion of the low-potential (LP) heine, the proposed site of peroxidation. Most of the increase in distortion occurs when high affinity Site ! is occupied by Ca 2~, with a small further increase upon filling Site II with Ca 2~ or Mg 2+. Changes in the heme substituent modes indicate that the macrocycle conformational change is associated with the Ca2~-dependent conformation of the covalently attached pentapeptide of the LP heme. Upon reduction of the high-potential heme and occupation of the Ca 2+ binding sites, structural changes of the peroxidatic heine are amplified, resulting in loss of the sixth ligand and peroxidase activity.

I. Pauleta S.R., Lu Y., Goodhew C.F., Moura I., Pettigrew G.W. and Shelnutt J.A. Biochemistry, 40, 6570-6579 (2001)

Sandia is a multiprogram laboratory operated by Sandia Corp., a Lockheed Martin Co., for U.S. Dept. of Energy under Contract DE-AC04-94AL85000. SRP thanks FCT-PRAXIS XXI (BD/18297/98) for financial support.

Journal of lnorganic Biochemistry 86 (2001) 387

Comparison of the structures of adducts of DNA-DNA cross-links formed by di and tri-nuclear platinum compounds

Y u n Q u ~, N e e l J. S c a r s d a l e b, M y - C h a u T r a n a, N i c h o l a s Far re l l a

Depar tmen t o f Chemis try , Virginia Commonweal th Universi ty . 1001 West Main st. Richmond, VA 23284. USA (e-mail: y q u @ v c u . e d u )

~ Depar tmen t o f B iochemis t ry , Virginia Commonweal th Universi ty , PO Box 980614, Richmond, VA 23284, USA

BBR3464 (1,0,1/t,t,t a Phase II anticancer drug) t and its dinuclear analog (1,1/t,t) form 1,4-GG and 1,6-interstrand cross-links with 8-mer and 10-mer oligonucleotides, with the two platinum atoms coordinated at the N7 positions of guanines on opposite DNA strands. These long-range interstrand cross-linked adducts have been characterized and analyzed by mass spectrometry and NMR spectroscopy. In the 1,4 cross-link the strong H(8)/HI' intraresidue crosspeaks for G3, A7 resonances are consistent with a syn-conformation for these bases. Comparison of the structures of the 1,4 cross-links formed by 1,0,1/t,t,t and 1,1/t,t show several interesting differences - a lack of severe DNA distortion in the 1,0,1/t,t,t-DNA adduct is evidenced from the permanence of base stacking and the integrity of the Watson-Crick base pairs. Further differences are seen in the "center" of the strands (the interior loop of two Pt binding sites) where the two G (G3 & O3') and T (T4, T4') imino protons have two distinct positions only in the 1,0,1/t,t,t-DNA adduct.

1. Farrell, N. Polynuclear Charged Platinum Compounds as a New Class

F OK /NH~ HN\ /el "7 2+ 1

T2 A , , ' H3N NH2(CH2)~H~N NH

G3 HC6 ' 1 ,lJt,[ i

. . . . .

qfAnticancer Agents. Toward a New Paradigm; Kelland, L. R., Farrell, N., Eds; Humana Press Inc.: Totowa, N J, 321- 338(2000)

This work was supported by operating grants from the American Cancer Society and National Institutes of Health.

EXAFS studies of mononuclear and dinuclear non-heme iron enzymes

L u c a O u a r o n i a, B r i a n J. B r a z e a u b, A m y M. R o c k l i n b, V i c t o r i a C o d r i n a P o p e s c u b, J o h n D. L i p s c o m b b,

L a w r e n c e Que , J r a.

~' Univers i ty o f Minneso ta , Depar tmen t o f Chemis try and Center f o r Metals in Biocatalys is , 207 "Pleasant St SE, Minneapol i s , MN 55455 (e-mail: quaron i@chem.umn.edu) . ~' Univers i ty o f Minnesota , Depar tmen t o f B iochemis t ry and Biophys ics and Center for Metals in Btocatalys ts , 3 i 2 Church Street , Minneapol is , MN 55455.

Extended X-ray Absorption Fine Structure (EXAFS) has been applied to address structural issues in the biochemistry of the non-heine iron enzymes aminocyclopropylcarboxylic acid oxidase (ACCO) and methane monooxygenase (MMO).

ACCO utilizes a mononuclear iron center to catalyze the synthesis of ethylene in plants, in a process that involves ACC, ascorbic acid and bicarbonate. The ligation of the iron center and its interaction with substrates and cofactors is still under debate. We use EXAFS to characterize the first coordination sphere of the metal and its changes upon binding of small molecules. We show that substrate binds directly to the iron, confirming previous studiesL Moreover, we show that bicarbonate binding to the enzyme does not affect the first coordination sphere of the metal.

MMO catalyzes the hydroxylation by dioxygen of a variety of small organic molecules, including simple alkanes and arenes. The enzyme/product complex is expected to provide insight into the mechanism of oxygen insertion into the substrate. The enzyme/product complex denominated intermediate T, detected during the oxidation of nitrobenzene 2, is believed to have the nitrophenol product bound to the diiron center in a bridging fashion. In this work we describe the complex of the enzyme with phenol, and discuss its validity as a model for intermediate T. 1. Rocklin, Amy M.; Tierney, David L.; Kofman, Victoria; Brunhuber, Norbert M. W.; Hoffman, Brian M.;

Christoffersen, Rolf E.; Reich, Norbert O.; Lipscomb, John D.; Que, Lawrence, Jr.; Proc. Natl. Acad. Sci. U. S. A. 96(14), 7905-7909 (1999).

2. Lee, Sang Kyn; Nesheim, Jeremy C.; Lipscomb, John D.; J. Biol. Chem. 268(29), 21569-21577 (1993).

388 Journal of Inorganic Biochemistry 86 (2001)

Mechanism of oxygen activation by o~-keto acid-dependent iron enzymes and their models

L a w r e n c e Que , Jr. a, R o b e r t P. H a u s i n g e r b, Er ic L. H e g g ~, R a y m o n d Y. N. H o a, A i m i n L iu a, M a r k P.

M e l m a, M a t t h e w J. R y l e b.

"Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA, (e-mail. que@chem.umn.edu) Departments of Microbiology & Molecular Genetics and Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA

c~-Ketoglutarate (c~-KG)-dependent dioxygenases constitute a large class of non-heme iron-containing enzymes that are essential for the biosynthesis of a diverse array of biological compounds, the catabolism of selected biomolecules and the sensing of oxygen in cells. The bidentate coordination of an a-keto acid to an iron(II) center via the keto group and the carboxylate gives rise to metal-to-ligand charge-transfer transitions. Resonance Raman studies of these transitions have allowed us to assign the vibrational features associated with this chromophore, t Exposure of one model compound to oxygen results in the hydroxylation of a ligand aryl ring. This mirrors the reactivity observed in the enzyme TfdA which also hydroxylates an aromatic residue in the active site in the absence of substrate, z Our spectroscopic and reactivity studies of these enzymes and model complexes provide useful insights into the mechanism of oxygen activation by cc-KG-dependent dioxygenases.

2 N ,o4 ........ ?

0.711

O.Io

O.26

O.N ~so 4oo 4so soo sso soo sso 700 150 soo

;,. ( n m ] t

1. HoR.Y.N. etal.,J. Am. Chem. Soc., 123,5022-5029(2001) 2. Liu A. et al., J. Am. Chem. Soc., 123, 5126 5127 (2001)

Interaction of platinum pyridin-2-yl-acetate complexes with DNA and protein models

S. M. O. Quintala~ Y. Qu b, H. I. S. Nogueira a and N. Farrell b a Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal, squintal@dq.ua.pt b Department of Chemistry, Virginia Commonwealth University, VA 23284-2006, USA

The therapeutic effects of platinum complexes are related to the formation of DNA coordination of platinum to the guanine N7 positions. Other biomolecules may bind to platinum, especially those having sulfur in their structure, for which platinum presents a high affinity. The platinum-sulfur interactions have traditionally been associated with undesired phenomena, such as resistance and toxicity. That leads to a strong interest in the study of the competitive interaction of platinum complexes with DNA and protein models. Here we report the reactions of trans-[PtCl(pyAc-N,O)(NH3)], trans-Pt, and its geometric isomer cis-[PtCl(pyAc-N,O)(NH3)], cis-Pt, (pyAc=pyridin-2-yl-acetate) with 5'-guanosine monophosphate, 5'-GMP, and N-acetyl-L-methionine, AcMet. These reactions were investigated by ~H and Z95pt NMR spectroscopy. The formation of trans-Pt-5'-GMP, cis-Pt- 5'-GMP, trans-Pt-AcMet, cis-Pt-AcMet, AcMet-Pt-AcMet adducts was observed. The 5'- GMP-Pt-5'-GMP adduct is detected only after long incubation times. AcMet-cis-Pt-5'- GMP species are detected in solution, during the reactions between cis-Pt-5'-GMP adduct AcMet adduct and 5'-GMP.

adducts through preferential

0 0

HN , ° ' S ' c . 2 3 ~p~ O H 2 CI N

trans-Pt cis-Pt

and AcMet or betwe6n cis-Pt-

1. U. Bierbach, and N. Farrell, J.B.I.C., 3,570, (1998).

S. M. O. Quintal thanks Funda~o para a Ci~ncia e a Tecnologia and Fundagao Caloust Gulbenkian, Portugal, for financial support.

Journal of lnorganic Biochemistry 86 (2001) 389

Spectroscopic characterization of the copper sites of Fet3p, a new member of the multicopper oxidases family

Li l i ana Q u i n t a n a r a, A m y E. P a l m e r a, T i m o t h y M a c h o n k i n a, Sco t t S e v e r a n c e b, T z u - P i n W a n g b, Dan i e l J. K o s m a n b, and E d w a r d I. S o l o m o n a

" Department o f Chemistry, S tanford University, Stanford, CA 9430, USA ( l i l ianaq@Stanford. edu) t, Department o f Biochemistry , School o f Medicine and Biomedical Sciences, State University o f

New York, Buffalo, NY 14214, USA

The multicopper oxidases are a class of enzymes that couple the four-electron reduction of dioxygen to water with the four sequential one-electron oxidations of substrate. The active site contains one type 1 or blue copper, one type 2 or normal copper, and one type 3 or coupled binuclear Cu site. Fet3p is a new member of the multicopper oxidase family that exhibits ferroxidase activity, and it is required for high affinity iron uptake in yeast. A recombinant form of Fet3p has been successfully expressed in Saccharomyces cerevisiae yeast I and a type 1 copper depleted form (T1D) has been generated, facilitating the spectroscopic characterization of the copper sites in this protein. The T1 Cu site has been characterized by EPR, CD, MCD, and resonance Raman spectroscopy; and its electronic structure and reactivity with different substrates have been compared to the T1 Cu sites in other multicopper oxidases 2. The trinuclear cluster in Fet3p has been characterized by EPR, CD, and MCD; and the effects o fpH and fluoride binding on the spectroscopic features of the type 2 and type 3 coppers have been studied. A comparison of the trmuclear cluster in the T ID Fet3p with that in the type 1 mercury-substituted derivative of Rhus vernicifera laccase will be presented.

1. Hassett, R. F.; Yuan, D. S.; Kosman, D. J., J. Biol. Chem., 273, 23274-23282 (1998) 2. Machonkin T.E.; Quintanar L.; Palmer A. E.; Hassett R.; Severance S.; Kosman D. J.; Solomon E. I., J. Amer. Chem

Soc., ASAP Article (2001) Acknowledgment: National Institute of Health DK31450 (to E.I.S.) and DK53820 (to D.J.K.)

Dinuclear iron(Ill) complexes as models of methane monooxygenaseNathalie R a f f a r d a, F r6d6r ic B a n s e a, M a r t i n e N i e r l i c h b, E l o d i e A n x o l a b 6 h ~ r e - M a l l a r t a, Ke i j i M i k i c, J ean -

J a c q u e s G i r e r d a.

Laboratoire de Chimie Inorganique, UMR CNRS 8613, Inst i tut de Chimie Moldculaire d'Orsay, Universit~ Paris-Sud, 91405 Orsay Cedex, France, (e-mail: natraf fard@icmo.u-psud. fr )

b C.E.A. S a c l a y , D R E C A M / S C M B a t 125, 91191 Gif-sur-Yvet te Cedex, France C Hydrocarbon Research Lab., Nat ional lns t i tu t for Resources and Environment , Tsukuba, Japan

Methanotrophic bacteria use methane as a source of energy and carbon. The first step of that process is methane oxidation in methanol by Methane MonoOxygenase (MMO) according to the reaction: CH4 + 02 + 2H ÷ + 2e "--) CH3OH + H20. The enzyme hydroxylase (MMOH) has been crystallized [11 and contains, at the native state, two ferric ions linked by carboxylato, oxo or hydroxo bridges. The models of the active site of MMO we synthesized are dinuclear iron(III) with a single oxo bridge: in view of industrial applications we used two simple amino- pyridine ligands. We observed alcane oxidation using 02 and electrons for one of the two complexes (that presented on the figure). Moreover, we studied the mononuclear iron(II) complexes with the same ligands and their reactivity with O2: the complex with the ligand which gives results in catalysis is precisely that which ta reacts with 02 to give the asymmetric dinuclear iron(III). We are now studying the stoichiometric oxidation of alcanes by 02 in presence of the mononuclear Fe(II) complexes.

1. Rosenzweig A.C.; Frederick C.A.; Lippard S.J.; Nordlund P., Nature, 366, 537-543 (1993).

390 Journal of lnorganic Biochemistry 86 (2001)

Metal lothionein and its active isoforms in human megakaryocytes

Mohammad Tariqur Rahman and Marc De Ley Laboratory for Biochemistry, Department of Chemistry, Katholieke Universiteit Leuven, Belgium

A search for the biological role of Cd in mammalian systems has ended up with the identification of a low Mr protein and termed metallothionein (MT) for its high metal and sulfur content 1. The existence of MT has been found to be crucial for the heavy metal homeostasis and detoxification. In humans MT is expressed in many cells/organs including RBC-', its precursors 3 and platelets 4 etc. Nucleated precursors like megakaryocytes (MK) were hypothesized 4 as the possible site of origin of MT in their terminally differentiated anucleated forms i.e. platelets, incapable of active protein biosynthesis. Here we report the in vitro biosynthesis of MT in MK. Megakaryocytes have been separated in considerable number (_>60%), as revealed by M~iy-Griinwald-Giemsa staining, in the positively selected CD61+ cells from human cord blood mononuclear cells, using magnetic cell sorting. In vitro upregulation and/or induction of total MT transcripts was observed in these cell populations at 48h post-treatment with 100~tM of Zn supplement. Upregulated expression of MT at the protein level was also detected by immuno-histochemical staining using monoclonal anti-MT (E9) in MK present in similar in vitro cultures of CD61 + cells. Nuclear localization of MT in some of the MK indicates its possible involvement in differentiation. Using RT-PCR, 7 out of 10 functional MT isoforms specific amplified retro-transcripts, e.g. MT-0 (MT-IH), MT-1A, MT-1B, MT-1E, MT-1G, MT-1X, MT-2A were detected in CD61~ cells just after the separation, hence considered as the active isoforms in those cell populations.

1. Margoshes M. and Vallee B.L., J. Am. Chem. Soc., 235, 3460-3465. (1957) 2. Grider A., Bailey L.B., Cousins R.J., Proc. Natl. Acad. Sci. USA., 87, 1259-1262. (1990) 3. Rahman M.T. and De Ley M., Eur. J. Biochem., 268, 849-56. (2001) 4. Sugiura T. and Nakamura H., Int. Arch. Allergy. Immunol., 103,341-348. (1994)

We acknowledge the Fonds voor Wetenschappelijk-Onderzoek-Vlaanderen (G.0410.98) for the research grant. MTR is supported by a KU Leuven scholarship. We wish to acknowledge the ICBIC10 organizer for the grant for MTR as young SBIC member to attend this conference.

Novel "Roussin esters" [Fe2(IA2-SR)2(NO)4] as NO donors: synthesis, structural and spectroscopic characterization

0. A. Rakova, Sanina N. A., Aldoshin S. M., Shulga Yu. M., Kulikov A. V. Institute of Problems of Chemical Physics RAS, 142432, Chernogolovka, Russian Federation (e-mail: rakova@icp.ac.ru)

Interest to synthesis and investigation of new "Roussin esters" is due to their possibly wide application as adjuvants of chemo-, radiotherapy and nitrovasodilators in medicine; as possible candidates for photochemical delivery of NO I and pharmacologically active thiols to biological targets; also as studying of Fe-S nitrosyl interaction with active substrates in vivo such as O2-" and OH'.

This work presents a synthesis, structural and spectroscopic investigation of novel [Fez(~t2-SR)2(NO)4] complexes where R are heterocycles containing one or more nitrogen atoms which are antibacterial agents, inhibitors of enzymes, antimetabolites. We found the formation of binuclear Fe-S nitrosyls generated from iron (II) sulfate, nitric oxide and heterothiols highly depends on pH, pK~ of thiols and medium polarity. Under the action of polar solvents the diamagnetic "Roussin esters" are readily converted into [Fe(SR)2(NOh]- complexes with EPR signals at g~2.032

When light with ~. = 254 nm was used the decomposition of binuclear structure of the complexes in solid state as well as the release of volatile gases: nitric oxide (m/z =30) and nitrous oxide (m/z = 44) were observed 3. The mechanism to explain the formation of nitrous oxide was proposed.

I. Lorkovic I., Bourassa J., Lee B., Kudo S., Ford P.C., et al., Coord. Chem. Rev., 171, P. 185-205 (1998). 2. Rakova O.A., Sanina N.A., Kulikov A.V., Shulga Yu.M., Aldoshin S.M., in Abstract of XI Int. Conf. "Magnetic

Resonance in Chemistry and Biology", Zvenigorod-2001, P. 162. 3. Rakova O.A., Sanina N.A., Shulga Yu.M., Martynenko V.M., Aldoshin S.M., Doclady Chemistry, (2001), in press.

This work has been supported by INTAS-99-00209