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Journal of Inorganic Biochemistry 86 (2001) 291 Interaction of non-heme ferrous active sties with substrates: structure/function correlations for phenylalanine hydroxylase and bleomycin Jyllian N. Kemslev a, Kelly Loeb Zaleski a, Nata~a Mitid b, Supratim Datta c, Tara Acharya b, John P. Caradonna c, and Edward I. Solomon ~ ~ Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA (e-mail. Kemsley@stanford. edu) ~ Department of Chemistry, Yale University, New Haven, CT06520, USA Department of Chemistry, Boston University, Boston, MA 02215, USA Mononuclear ferrous sites catalyze a variety of reactions involving the binding and activation of dioxygen. A general mechanism has been proposed in which substrate binding to the active site activates the iron atom for reaction with dioxygen. ~ Our studies on two such systems, phenylalanine hydroxylase (PAH) and bleomcyin (BLM), are extended to elucidate this mechanism and examine its biological implications. PAH catalyzes the coupled hydroxylation of L-Phe and tetrahydropterin to generate k-Tyr and 4a-hydroxytetrahydropterin. Dysfunction of this enzyme in humans results in the disease phenylketonuria. We have used CD and MCD to demonstrate that binding of both L-Phe and pterin to the enzyme results in the opening of a coordination position on the ferrous site, directly implicating the iron in the coupled hydroxylation. These studies are now expanded to include disease-inducing enzyme mutants in order to understand the disease mechanism at a molecular level. BLM is a glycopeptide antibiotic used in cancer chemotherapy to seIectively cleave DNA. Our previous work has demonstrated that the ferrous active site is six-coordinate and provided insight into the metal ligands. Current studies focus on the interaction of the iron site with DNA oligomers and indicates that binding of this substrate results in significant changes to the metal environment that are dependent on DNA strand composition, correlating with the preferred cleavage sites. ~. Solomon, E.I. et al., Chem. Rev., 100, 235-349 (2000). NIH (EIS GM40392, JPC GM61208) The structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium Arthrospira maxima Cheryl A. Kerfeld a, Michael R. Sawaya a, David W. Krogmann, b Kwok K. Ho, b and Todd O. Yeates a ~'Molecular Biology Institute, UCLA, Los Angles California (e-mail: [email protected]) #Department of Biochemistry, Purdue University, West Lafeyette, Indiana Cytochrome c6 and cytochrome c-549 are small (89 and 130 amino acids, respectively) monoheme cytochromes that function in photosynthesis. They appear to have descended relatively recently from the same ancestral gene but have diverged to carry out very different functional roles, underscored by the large difference between their midpoint potentials of nearly 600 mV. We have determined the X-ray crystal structures of both proteins isolated from the cyanobacterium Arthrospira maxima. The two structures are remarkably similar, superimposing on alpha carbon atoms with an rmsd of 0.75 A. Comparison of the two structures offers insights into the structural determinants of midpoint potential in the two proteins. In addition, the crystal packing of both A. maxima cytochrome c-549 and cytochrome c6 suggests the proteins oligomerize. In one crystal form of cytochrome c6, 24 molecules are arranged in a hollow shell with approximate 432 symmetry. Putative functions of the oligomerization state observed in the crystals will be discussed. Finally, the cytochrome c-549 dimer we observe can be readily fit into the recently described model of cyanobacterial Photosystem II. The United States Department of Agriculture is acknowledged for its financial support of this work.

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Journal of Inorganic Biochemistry 86 (2001) 291

Interaction of non-heme ferrous active sties with substrates: structure/function correlations for phenylalanine hydroxylase and bleomycin

Jy l l i an N. K e m s l e v a, K e l l y L o e b Za l e sk i a, Na t a~a Mit id b, S u p r a t i m D a t t a c, T a r a A c h a r y a b, John P.

C a r a d o n n a c, and E d w a r d I. S o l o m o n ~

~ Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA (e-mail. Kemsley@stanford. edu) ~ Department of Chemistry, Yale University, New Haven, CT06520, USA

Department of Chemistry, Boston University, Boston, MA 02215, USA

Mononuclear ferrous sites catalyze a variety of reactions involving the binding and activation of dioxygen. A general mechanism has been proposed in which substrate binding to the active site activates the iron atom for reaction with dioxygen. ~ Our studies on two such systems, phenylalanine hydroxylase (PAH) and bleomcyin (BLM), are extended to elucidate this mechanism and examine its biological implications. PAH catalyzes the coupled hydroxylation of L-Phe and tetrahydropterin to generate k-Tyr and 4a-hydroxytetrahydropterin. Dysfunction of this enzyme in humans results in the disease phenylketonuria. We have used CD and MCD to demonstrate that binding of both L-Phe and pterin to the enzyme results in the opening of a coordination position on the ferrous site, directly implicating the iron in the coupled hydroxylation. These studies are now expanded to include disease-inducing enzyme mutants in order to understand the disease mechanism at a molecular level. BLM is a glycopeptide antibiotic used in cancer chemotherapy to seIectively cleave DNA. Our previous work has demonstrated that the ferrous active site is six-coordinate and provided insight into the metal ligands. Current studies focus on the interaction of the iron site with DNA oligomers and indicates that binding of this substrate results in significant changes to the metal environment that are dependent on DNA strand composition, correlating with the preferred cleavage sites.

~. Solomon, E.I. et al., Chem. Rev., 100, 235-349 (2000).

NIH (EIS GM40392, JPC GM61208)

The structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium Arthrospira maxima

C h e r y l A. K e r f e l d a, M i c h a e l R. S a w a y a a, D a v i d W. K r o g m a n n , b K w o k K. H o , b and T o d d O. Y e a t e s a

~'Molecular Biology Institute, UCLA, Los Angles California (e-mail: k e r f e l d @ m b i . u c l a . e d u ) #Department of Biochemistry, Purdue University, West Lafeyette, Indiana

Cytochrome c6 and cytochrome c-549 are small (89 and 130 amino acids, respectively) monoheme cytochromes that function in photosynthesis. They appear to have descended relatively recently from the same ancestral gene but have diverged to carry out very different functional roles, underscored by the large difference between their midpoint potentials of nearly 600 mV. We have determined the X-ray crystal structures of both proteins isolated from the cyanobacterium Arthrospira maxima. The two structures are remarkably similar, superimposing on alpha carbon atoms with an rmsd of 0.75 A. Comparison of the two structures offers insights into the structural determinants of midpoint potential in the two proteins.

In addition, the crystal packing of both A. maxima cytochrome c-549 and cytochrome c6 suggests the proteins oligomerize. In one crystal form of cytochrome c6, 24 molecules are arranged in a hollow shell with approximate 432 symmetry. Putative functions of the oligomerization state observed in the crystals will be discussed. Finally, the cytochrome c-549 dimer we observe can be readily fit into the recently described model of cyanobacterial Photosystem II.

The United States Department of Agriculture is acknowledged for its financial support of this work.

292 Journal of Inorganic Biochemistry 86 (2001)

Chemical reactivity of dinuclear complexes with hydrophobic binding pockets

Berthold Kersting

Institut fiir Anorganische und Analytische Chemic, Universitiit Freiburg, Albertstr. 21, D-79104 Freiburg, GERMAN Y (e-mail. kerstber@sun2, ruf uni-freiburg, de)

The active site of many metalloproteins is located in a hydrophobic environment in the center of the peptide matrix.t The specific microenvironment about the reaction center drastically alters the chemical reactivity, as is seen, for example, in Cytochrome P450. The development of transition metal complexes with hydrophobic binding pockets is therefore of great interest. We have begun to explore the chemical reactivity of binuclear complexes with hydrophobic binding pockets. For the preparation of such complexes we use macrocyclic amine-thiophenolate ligands. By varying the substituents R (R = H, Me, Et, Pr) it is possible to control the size and shape of the binding pocket around the free coordination site in complexes of type A. There is a clear effect of the choice of R on the chemical reactivity. We will report on our findings.

1. S, J. Lippard, J. M. Berg, Bioanorganische Chemie, Spektrum Akademischer Verlag, Heidelberg, 1995.

-7 n+

R R

c. .2- . / / ~---.r~\\.~ . 7

R R

A

Financial support of this work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

Reactivity of a binuclear iron (III) complex toward hydrogen peroxide

Renata C. K. Kaminski a and Sueli M. Drechsel a

a Department of Chemistry, Federal University ofParanf ,Jardim das AmOricas s/n, zip. 81531- 990, P.O. 19081, Curitiba, Brazil. (e-mail: [email protected], br)

Recently some studies have shown that well designed binuclear iron complexes were efficient catalysts for the oxidation of hydrocarbons, providing an original strategy in the search for new catalysts. During the past decade, several reports have discussed the iron-induced activation of i i l ~ ~ hydrogen peroxide (H202) for the catalytic and selective oxygenation of hydrocarbon substrates. It has been suggested that the catalytic activity of these systems depends on ~" the ability of the iron sites to bind the peroxide and generate oxidizing high-valence iron i species 1. The reaction of a methanol solution of Hbpa ((2-hydroxybenzyl)(2- _~ methylpyridyl)amine) with FeC13.6H20 and triethylamine (in 3:2:1/2 proportion) produced dark blue crystalls (1). The reactivity of 1 towards H202 was accompanied in acetonitrile solution to study the possible catalytic activity of the complex in oxidation of organic substrates by hydrogen peroxide. The addition of peroxide in a proportion of 1:4 (1 :H202) results in a green solution with ~,m.e, in 672 nm (Figure). Similar spectra were observed for g-peroxo - diiron(III) complexes 4. The cyclic voltammetry of the green solution shows two irreversible waves (E~ = - 0.3 V e E2 = - 0.8 V vs Ag/AgC1). The reduction processes are cathodic dislocated in comparison of the observed peaks of 1. After 5 minutes, the peak current has decayed by about 50% indicating the instability of the green specie. Despite the green specie has been observed, catalytic tests with this compound in alkane and alkenes oxidation, as yet, has produced small yields, next to 5,5% to ciclohexanol.

1. M6nage, S.; Wilkinson, E. C.; Que, L. Jr; Fontecave, M.. Angew. Chem. Int. Ed. Engl., 34) 203-205 (1995.

We aknoledee UFPR, CNPa. LACTEC. FUNPAR. PADCT.

Journal of Inorganic Biochemistry 86 (2001) 293

Synthesis of vanadyl and zinc(II) complexes of N-substituted 3-hydroxy- 4(1H)-pyridinethiones and their insulin-mimetic activities

Akira Ka toh a, Takesh i T s u k a h a r a a, R y o t a Saito a, Yu taka Y o s h i k a w a b, Yosh i t ane K o j i m a b, and H i r o m u Sakura i c

~Department of Applied Chemistry, Faculty of Engineering, Seikei University, Musashino-shi, Tokyo 180-8633, Japan, bGraduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan, CDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Yamasina-ku, Kyoto 607-8414, Japan

At the present time, diabetes mellitus afflicts over 140 million people in the world. Recently, vanadyl and zinc(II) complexes have been demonstrated to have high insulin-mimetic activities. ~'2 As an extensive study on application of heterocycles to chemotherapeutic agents, we describe here synthesis of vanadyl and zinc(II) complexes of N-substituted 3-hydroxy-4(1H)-pyridinethiones and their insulin-mimetic activities. N-Substituted 3-hydroxy-4(1H)-pyridinethiones (2a,b) were prepared from maltol(1) via 2 or 3 steps. Vanadyl-(3a,b) and zinc(II) complexes(4a,b) were synthesized from tretrnent of 2a,b with V O S O 4 and ZnSO4 ill aqueous solutions, respectively. The insulin-mimetic activity of the complexs was evaluated by in vitro experiments, in which the inhibition of release of free fatty acid (FFA) from isolated rat adipocytes treated with epinephrin was estimated. Vanadyl complexes(3a,b) showed moderate activities (IC5o: 11.16 mM for 3a; 2.45 mM for 3b). Zinc(II) complex(4a) colud not be measured owing to its insolubility, while zinc(II) complex (4b) showed extremely high activity (0.044 mM when IC50 of VOSO4 was calibrated to be 1.0 mM). We are under investigation whether this activity virtually arises from the complex or from a result of its anti-seborrheial activity. I. A. Katoh, K. Taguchi, Y. Fujisawa, T. Takino, and H. Sakurai, Chem. Lett., 866-867(2000). 2. Y. Yoshikawa, K. Kawabe, H. Miyake, H. Sakurai, and Y. Kojima, Chem. Lett., 874-875(2000).

Me /

_S O ..L R i / / ~ " ' . M / Y / ' N "

Me

3a, b : M = V = O a: R = M e

4a, b ; M = Zn b: R=Ph

294 Journal of lnorganic Biochemistry 86 (2001)

Dioxygen reactivity of reduced heme-copper complexes utilizing tetraarylporphyrinates tethered with a tridentate ligand

Eunsuk Kim, Marie-Aude Kopf, Kenneth D. Karlin Department of Chemistry, Johns Hopkins University, Baltimore, MD, 21218, USA

In our continuing interest in developing synthetic models for the heme a3-Cu B O2-binding, O2-reduction, and proton pumping site in cytochrome c oxidase, we have prepared the heterobinuclear complexes [(2L)FeHCul]~ (1), and [(4L)FenCuil+ (2), in which a Cu I moiety with tridentate ligand bis(2-pyridylethyl)amine is tethered to an FeH- tetraarylporphyrin with or without a covalently attached pyridine axial base. Exposure of complexes 1 and 2 to 02 yields low temperature stable peroxo complexes [(2L)Fem-O2-CuH] + (3), and [(4L)FeHI-o2-Cun] + (4), which decompose to give g-oxo bridged complexes, [(2L)Fem-O-CuH] + (5), and [(4L)Feln-O-Cun]+ (6), at room temperature. Characterization of complexes 1- 6 including resonance Raman, UV-Vis, NMR, and EPR spectroscopies, and dioxygen uptake titration will be discussed.

1. Marie-Aude Kopf and Kenneth D. Karlin., Inorg. Chem,, 38, 4922-4923 (1999)

The National Institutes of Health (USA) is gratefully acknowledged for financial support.

New thiyl and phenoxyl radical complexes

Shuji Kimura, Eberhard Bothe, Eckhard Bill, Thomas Weyhermfiller, Karl Wieghardt. Max-Planck-Institut fiir Strahlenchemie, Stiftstr. 34-36, D-45470, Miilheim an der Ruhr, Germany (e-mail.kimura@mpi-muelheim. mpg. de)

The study of metalloproteins containing amino acid based radicals has enormously expanded in recent years.~ In some of these proteins, the intermediate formation of phenoxyl radicals has been spectroscopically well established. In contrast, although thiyl radicals have been invoked in some catalytic cycles, their electronic structure is less well characterized.

We have designed and synthesized new asymmetrically N-substituted 1,4,7- triazacyclononane-l,4-dicarboxylates which contain a thiophenolate or phenolate pendent arm in order to synthesize mononuclear complexes and to characterize their oxidized forms.

With trivalent metal ions these ligands form very stable neutral mononuclear complexes (MmL l, M=Fe, Co, Ga, Ru, Rh; MmL 2, M=Co, Ga). Cyclic voltammetry shows that all five ML t complexes undergo a reversible, ligand- based, one-electron oxidation generating the monocations [MmL~'] * which contain a coordinated phenoxyl radical. [CoHIL 2] in CH2C12 at -20 °C displays a reversible one-electron oxidation yielding a [CoraL2"] +. Its electronic spectrum shows intense transitions at 509 nm (~=2600 M l c m -t) and 784 nm (1030 M-lcml). The EPR spectrum of this species at 90 K proves the presence of a coordinated thiyl radical (gi~o=2.0226; Ai~o(59Co)=10.7 G).

I. Stubbe, J. etal. Chem. Rev. 1998, 98, 705.

o o

X=O [L 1 ]3-

X=S [L2] 3"

Fig. 1. Ligands.

Journal of Inorganic Biochemistry 86 (2001) 295

Effect of exogenous metal ions on the inhibition of trypsin by diamidines

Sandra R. Kircus, H. Hol den Thorp

Department of Chemistry, University of North Carolina at Chapel Hill, Kenan Labs, Chapel Hill, NC 27599, USA (e-mail. kircus@email, unc.edu)

Aromatic diamidines are modest inhibitors of several serine protease enzymes (1). However, incorporating an exogenous metal ion into the binding site, which does not normally coordinate a metal ion, can increase the affinity of the small molecule diamidine by several orders of magnitude (2,3). We have studied Zn 2", Cd 2+, and Mn 2. mediation of diamidine binding to the serine proteases bovine trypsin and bovine thrombin. Each particular system affords inhibition to varying degrees, even within the same inhibitor/metal ion pair but differing protease.

1 Geratz, J.D.; Tidwell, R.R.J . Med. Chem. 1976, 19, 634-639. 2. Katz, B.A.; Finer-Moore, J.S.; Jenkins, T.E.; Johnson, C.R.; Ross, M.J.; Luong, C.; Stroud, R.M.

391,608-612. 3. Thorp, H.tt. Chem. AndBiol. 1998, 5, R125-127.

Nature 1998,

We thank Professor R. Tidwell for gift of the inhibitor.

Covalency contributions to reactivity in xanthine oxidase and [MoOS] + models

Mart in L. K i rk a, N i c k D. Rub ie a, Kat r ina Pear iso ~, Chris t ian D o o n a n b, G r a h a m N. Geo rge c, and Char les G. Y o u n g b.

"Department of Chemistry, University of New Mexico, Albuquerque, NM87131-1096, USA (e-mail mkirk@unm, edu), School of Chemistry, University of Melbourne, Victoria 3010, NM 87131-1096, AUSTRALIA,

:Stanford Synchrotron Radiation Laboratory,Stanford, CA USA,

Members of the xanthine oxidase (XO) family of molybdenum enzymes are unique in that they function as true hydroxylases, formally incorporating an oxygen atom from water into a substrate C-H bond ~. Considerable effort has been put forth to understand the key components of the XO mechanism. A true catalytic intermediate, termed 'very- rapid', is formed during the course of reaction with xanthine and certain other substrates. At present there are two major proposals concerning the structure of this intermediate, and these relate to very different reaction mechanisms. 2'3 We have undertaken a comprehensive spectroscopic and electronic structure study of model systems that incorporate the key [MoOS] unit of the XO active site 4, as well as MCD and electronic structure studies of the enzyme 'very rapid' intermediate 5. The results are discussed in terms of the unique role of the Mo=S ~" orbital in catalysis.

1. Hille, R, Chem. Rev.,1996, 96, 2757-2816. 2. Palanichamy, M.; Choi, E.-Y.; Hille, R.; Hoffman, B.M.J. Am. Chem. Soc. 2001, 123, 2658-2663. 3. Canne, C.; Lowe, D.J.; Fetzner, S.; Adams, B.; Smith, A.T.; Kappl, R.; Bray, R.C.; Huettermann, J. Biochemistry

1999, 38, 14077-14087. 4. Rubie, N.D.; Doonan, C.; Peariso, K.; George, G.N.; Young, C.G.; Kirk, M.L. manuscript in preparation. 5. Jones, R.M.; Inscore, F.; Hille, R.; Kirk, M.L. Inorg. Chem. 1999, 38, 4963-4970.

296 Journal of Inorganic Biochemistry 86 (2001)

Reaction of carboplatin with methionine- and histidine-containing peptides

Marco Kleine. William S. Sheldrick Lehrs tuhl f i ir Ana ly t i sche Chemie, Ruhr-Univers i t iH Bochum, D-44780 Bochum, Germany

The second generation antitumor drug carboplatin [Pt{C6H4(COO)2}(NH3)2] ([C6H4(COO)212- = cyclobutane- 1, l-dicarboxylate) is less toxic than cisplatin and lacks its significant nephrotoxicity. A previous study of the reaction of the model compound [Pt{CH3CH(COO)2}(en)] with Ac-Met-OH and H-Gly-Met-OH demonstrated that I!S ring- opened complexes containing a monodentate dicarboxylate ligand are remarkably stable at neutral pill. This suggests that such thioether binding could play a role in carboplatin activation and prompted our present pH and time dependent HPLC and NMR investigations to allow a full characterisation of the reaction products for carboplatin itself with the tripeptides H-Gly-Gly-Met-OH, Ac-Gly-Gly-Met-OH and H-His-Gly-Met-OH. In analogy to cis-[PtC12(NH3)2], at low pH (3.3) rapid formation of the ~2Nmet, S six-membered chelate is followed by ammonia release and subsequent cleavage of the peptide backbone at the second metallated amide nitrogen Na in the resulting tridentate ~_3Na,Nmet,S species. In contrast, the relative stability of the initial ring-opened ~ S complex at neutral pH leads to preferred formation of a bifunctional 52S,S' adduct, for which trans ammine labilization facilitates an alternative reaction pathway to tridentate and cleaved complexes. The significance of competitive E~2N(imidazole),Smet macrochelation and/or purine N7-coordination in the presence of guanine nucleotides under such reaction conditions has been investigated.

1. auo Z., Hambley T.W., del Socorro Murdoch P., Sadler P.J. and Frey U., J. Chem. Soc., Dalton Trans., 469 - 478 (1997).

2. Hahn M., Kleine M. and Sheldrick W. S., JBIC, 6 (2001), online 28.4.01.

Characterization of three laecases from Marasmius quercophilus C30

Agnieszka Klonowska a, Christian Gaudin b, Marcel Asso c, Andr6 Foumel c, Thierry Tron a a Labora to i re de B io inorgan ique Structurale , UMR CNRS 651 7, case 452, Universi t~ d 'A ix -

Marse i l l e 111, Escadr i l l e Normand ie Niemen, 13397, Marsei l le , France (e-mail: a. k lonowska@lbs , u-3mrs. fr )

b Laborato ire d ' E c o l o g i e Microb ienne , Universi t~ d 'A i x -Marse i l l e HI, Marsei l le , France c BioOnergOtique et IngOnierie des Proteines , UPR, Marsei l le , France

The basidiomycete Marasmius quercophilus C30 produces at least four different extracellular laccase isoenzyrnes LACI, LACB, LACC and LAC21 when cultivated in the presence of copper andp-hydroxybenzoic acid. These enzymes, as revealed by the Southern blot analysis, are encoded by a gene family. We have already cloned seven members of this family among which, lacl, the structural gene coding for the major constitutive laccase (LAC 1)2 and, lac2, coding for the most active induced laccase (LAC2) 3 were formally identified. The heterologous expression (in S. cerevisiae) of a cDNA allowed us to produce a recombinant laccase whose fungal equivalent has not yet been detected. The three laccases characterized so far LAC1, LAC2 and LAC3, in spite of their relatively high homology (app. 80%) significantly differ by their catalytic properties. These differences will be discussed in the light of the 3D structure of the laccase from C. cinereus.

1. Klonowska A., Le Petit J. and Tron T., FEMS Letters., 2001, in press 2. Dedeyan B., Klonowska A., Tagger S., Tron T., Iacazio G., Gil G. and Le Petit J., Appl. Environm. Microbiol., 66,

925-929 (2000) 3. Klonowska A., Gaudin C., Foumel A., Asso M., Le Petit J. and Tron T., 2001, in preparation

The French Agency for Environment and Energy Management (ADEME) is acknowledged for a doctoral fellowship to A. Klonowska.

Journal of Inorganic Biochemistry 86 (2001) 297

Methane activation on Methane Mono-Oxygenase mimics

Peter-Paul H. J. M. Knops-Gerrits a, Atsushi Fukuoka b, William A. Goddard III c. D @ a r t e m e n t de Chimie, Univers iM Cathol ique de Louvain (UCL), Ba t iment LAVOISIER, Place L. Pas teur n° l , B-1348 Louvain- la-Neuve , Belgium T~l : +32-10-47 29 39, Fax ." +32-10-47 23 30," [email protected] l , ac. be

t, Catalysis Research Center, Hokkaido Universi ty , Sapporo 060, Hokkaido, Japan, c MPSC, Beckman Ins t i tu te (139-74) ,CalTech , Pasadena CA 91125, USA

Methane Mono-Oxygenase (MMO) is a di-iron active site containing enzyme that catalyzes the dissociative binding of molecular oxygen. To mimic the MMO active site we chose to study the heptapodate coordinated binuclear iron (II or III)-complexes of N,N,N',N'- tetrakis(2-benzimidazolylmethyl)-2-hydroxy- 1,3-diamino-propane (HPTB), N,N,N',N',- Tetrakis(2-pyridylmethyl)-2-hydroxy-l,3-diamino-propane (HPTP) in catalytic experiments and their finite cluster model N,N,N',N',-Tetrakis(2- iminomethyl)-2-hydroxy-l,3-diammo-propane (HPTM) in theoretical calculations. These have active sites of the form [Fe2(HPTL)(g-OH)]4+ or 2+. Quantum Mechanic structures are compared with experimental Mfssbauer, DRS, Magnetic, IR, Raman & EXAFS data. For the 02 binding on the reduced active site the g- q 1 :rl 1-O2 mode seems to preceed formation of the O=Fe-O-Fe=O bis-ferryl active site that reacts exothermally with methane by 50.56 kcal/mol. After the H abstraction from methane, the methyl radical recombines with the FeO center via a weak Fe-OCH3 bond in this model & with the loss of methanol FeIII-O-FeIII-is formed which is reduced to the Fe(lI,II) form. Ferryl groups are reactive two center three electron bonds.

H

P.P. Knops-Gerrits, P.A. Jacobs, A. Fukuoka, M. Ichikawa, F. Faglioni, & W.A. Goddard III, Reactivity of methane mono-oxygenase, insights from quantum mechanic studies on synthetic iron model complexes, J.Mol.Cat., A., 166, 3-15, (2001), Special Issue, P.P. Knops-Gerrits, M. Witko, R. Millini & W.A. Goddard III, Eds.

Thermally stable peroxodiiron(III) complex of hexapyridine ligand: synthesis, characterization, and activation.

Masahito Kodera, Motohiro Itoh, and Koji Kano. Depar tmen t o f Mo lecu lar Sc ience and Technology, Doshisha Universi ty , Kyo tanabe Kyoto ,610-

0321,Japan (e-mail." mkodera@mai l , doshisha .ac . jp)

A thermally stable (g-acetato)(g-oxo)(g-l,2-peroxo)diiron(III) complex [Fe2(O2)(O)(OAc)(L)](CF3SO3) (1) {L = 1,2-bis[2-(bis(2-pyridyl)methyl)-6-pyridyl]ethane} has been isolated from the reaction of [Fe2(O)(OAc)2(L)](CF3SO3)2 with H202 in MeCN in the presence of Et3N. The elemental analysis of 1 agrees with the formula [Fe2(O2)(O)(OAc)(L)] (CF3SO3)'5H20. A parent peak appears at m/z 739 ([Fe2(O2)(O)(OAc)(L)] +) in the FAB MS spectrum of 1. The UV-vis spectrum o f l shows two absorption bands at 510 (1300 M 1 cm 1) and 605 run (1310 M- i cm-i) characteristic of the (g-oxo)(g-l,2-peroxo)diiron(III) core. The resonance ~ ~ ' N ~ Raman spectrum of 1 shows two bands at 816 and 472 cm -~ which shift to 771 and 455 cm -1 by 1802-1abeling with H2~802, and are respectively assigned to the Vo.o and the VF,_ o of a bound peroxide. The Vo.o value of 1 is notably below the range of 848-900 cm -~ reported for a lot of (g-l,2-peroxo)diiron(III) complexes. The M6ssbauer spectrum of 1 at 4.2 K displays only a quadrupole doublet (AEQ = 1.67(8) mm/s and 6 = 0.53(8) CFsSO3 ram/s), indicating the high-spin diiron(III) unit is symmetrically bridged by the peroxide. Exchange coupling constant J value in 1 is -60 cm l , larger than -33 cm "l in [Fe2(O2)(OBz){HB(pz')}]. This is due to g-oxo bridge of 1. All these data demonstrate that 1 has a (g-acetato)(g-oxo)(g-l,2-peroxo) diiron(III) core structure, quite unique in the peroxodiiron(III) complexes known so far. The spontaneous decomposition of 1 in MeCN monitored at 605 nm obeys good first-order kinetics with k = 2.5 x 10 .5 s ~ at 300 K (the half-life time xl/z = 7.7 h), demonstrating that 1 is thermally stable. Upon addition of RCOC1 (R = m-C1C6H4, CH3, and CC13) in the presence of DMF (or DMA), 1 is activated to monooxygenate cyclohexane and cyclohexene to the corresponding alcohols and ketones.

298 Journal of lnorganic Biochemistry 86 (2001)

Metal ion release mechanism of the blue copper protein pseudoazurin

T a k a m i t s u K o h z u m a a, T o m o t a k e N i i z e k i a, K a t s u k o Sa to b, C h r i s t o p h e r D e n n i s o n b, S h i g e n o r i

N a g a t o m o c, a n d T e i z o K i t a g a w a c

~Department of Materials and Biological Sciences, Ibaraki University, Mito, Ibaraki 310-8512, JAPAN (e-mail: kohzuma@biomoI, sci. ibaraki.ac.jp)

bDepartment of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RU, UK

~Ii~stitute for Molecular Science, Okazaki, Aicki444-8585, JAPAN

Pseudoazurin is a type 1 blue copper protein which functions as the electron donor to a copper-containing nitrite reductase. The X-ray crystallographic structure ofAchromobacter o, cloclastes pseudoazurin shows that the protein is an eight stranded B-sandwich. The copper center is coordinated by two histidines (H40 and H81), a cysteine (C78), and a methionine (M86) in a distorted tetrahedral geometry. The copper ion in pseudoazurin can easily be substituted with other metal ions, as is the case in many blue copper proteins, but the mechanism of uptake and release of metal ions has not been clearly demonstrated. We have studied the metal ion release mechanism of Zn 2* and Cd 2+ substituted pseudoazurin through 235 nm excited UV resonance Raman (UVRR) spectroscopy. Zn 2+ and Cd 2" pseudoazurin give characteristic Raman bands for a metal ion coordinated imidazole at 1388 and 1384 cm 4 respectively. The Raman intensity of the histidine imidazole band diminishes in Zn 2+ or Cd > pseudoazurin upon lowering the pH, and an imidazolium band at 1408 cm 4 appears at pH < 4.0. The X-Pro bands at 1467 cm 4 in the metal-substituted pseudoazurins give a very similar pH dependence as apo-pseudoazurin. These findings suggest that the metal ion is released from the active site ofpseudoazurin at acidic pH accompanied by a change in the protein's secondary structure.

Dinuclear copper(ll) complex with a novel 30-membered octaazamacrocyclic ligand L, crystal structure, antitumor activities and cleavage to DNA

D e y u a n K o n g , A r t h u r . E. M a r t e l l , R a m u n a s . J. M o t e k a i t i s

Department of Chemistry, TEXAS A&M University, TX 77842-3012, College Station, USA (e-maii.-kong@mail. chem. tamu. edu)

A novel 30-membered macrocycle ligand with two phenolic pendent groups L (BDPH, 4, 6, 9, 12, 20, 23, 26, 29- octaaza-35, 36-dihydroxy- 16, 33-dimethyl-tricyclo[29.3.1.1,4,1S]hexatriaconta - 1 (35), 18,(36), 15,17, 31,33-hexaene) has been synthesized by the NaBH4 reduction of the Schiff base obtained from the [2+2] condensation between triethyltetraamine and diformyl-p-cresol. The dinuclear copper complexes also have been synthesized and structurally characterized with x-ray. [Cu2C30Hs0NsO2](CIO4)2.CH3OHo2H20 crystallizes in the monoclinic system, P2(1)/n space group, with a = 7.9662(7), b = 45.925(4), c = 11.0614(10) A, c, = 90 [3 = 105.187(4) y = 90 °, V = 3905.5(6) A 3, and Z = 4. Each copper ion was coordinated to four nitrogen atoms and one phenolic oxygen atom to form a pyramidal polyhedron. The protonation constants of BDPH and stability constants of copper complexes in 1:1 and 1:2 systems (BDPH:copper) were determined by potentiometric measurements at 25°C and # - 0.1 M KC1. Preliminary pharmacological experimental results showed that this copper complex have antitumor activities in vitro. And also ~,Nl~,o this complex can cleave the supercoiled pBR 322 DNA in the presence of 2 g H202.

1. Kong D. Y., Qin C., Meng L. H., and Xie Y. Y. Bioorg. Med. Chem. Lett. 9(8), 1087-1092 (1999) 2. A.E. Martell, R. J. Motekaitis, The Determination and Use of Stability Constants, 2nd ed., VCH Publishers: New

York, 1992

The Robert A. Welch Foundation is acknowledged for financial support.

cE28) C~13~

- cl~g~ cr14)

CI30I C1151 :1111

O¢;t

~) N21

Journal of lnorganic Biochemistry 86 (2001) 299

The macrocyclic compounds of ytterbium and neodymium as perspective luminescent labels for near-infrared spectral region

Yurii Korovin, Victor Kuz'min A. V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, 86 Lustdorfskaya doroga, 65080 Odessa, UKRAINE (e-mail. [email protected]

The increase of efficiency of 4f-luminescence of lanthanide ions, in particular Yb(III) and Nd(III), which observed in the near-IR spectrai region (the main emission bands at 980 and 1060 nm, respectively), is very important condition for use of this luminescence at the decision of bio-medical tasks. Now it can be stated, that the most high quantum yields of Ln(III)-luminescence are realized in their complexes with macrocyclic ligands modified chromophore-substituents that play the role of energy accumulator ("photoantenna"). ~ Thus, for the 12-membered azamacrocyclic (on the base of 2,6- substituted pyridine) complexes of where the chromophores are derivatives of xylenol orange, phthalexone S, methyltymol blue the luminescence lifetimes reached up 15 and 3 microseconds (for Yb-and Nd-complexes, respectively). The absorption, excitation and luminescence spectra were investigated for all the complexes. In addition of emission center design, and to better understanding the interactions of Ln-complexes with some of bio-organic molecules, we would like to present our results on the development of force field parameters for molecular mechanics studies of these complexes. Particular interest is caused by the influence of type photo-antenna-substituents on the conformational preference of conjugated and non-conjugated complexes. The parametefization is being developed through a combination of structural characterization of the key complexes by dynamic NMR spectroscopy, luminescent spectroscopy, molecular mechanics calculations and topological approach. The results show that some of these complexes are very suitable for application in bio-medical investigations.

1. Shevchuk S., Rusakova N., Turianskaya A.,Korovin Yu., Nazarenko N. and Gren A., J.Fluorescence, 8,225-228 (i1998)

The National Academy of Sciences of Ukraine is acknowledged for financial support.

Fullerene-stimulated penetration of bivalent metal ions through phosphatidylcholine liposomes

R. A. K o t e l n i k o v a a, G. N. B o g d a n o v a, G. V. Zo t ina a, V. S. R o m a n o v a b, Z. N. P a m e s b, A. I. K o t e l n i k o v a

alnstitute of Problems o f Chemical Physics RAS, Chernogolovka, Moscow region 142432, Russia (e-mail: kotel@icp, ac. ru) blnstitute of Organoelement Compounds RAS, Moscow, 117813, Russia

A significant question in studies of biologic activity of novel organic compounds is the understanding of the mechanism of their permeability through biologic membranes. The effect of water soluble amino acid derivatives of fullerene C60-A1 and C60-A1-A1 (C60-AD) have been studied on the permeability of model liposomes to Co z+. Procedure have been developed for investigation of permeability of chemical agents through lipid membranes, using a triplet probes method. Co -'+ ions act as quenchers of an exited triplet erythrosine. Upon an ultrasonic treatment of a suspension of phosphatidylholine with erythrosine it got embedded into the external and internal surfaces of the phospholipid membrane. The addition of Co 2" ions decreased the erythrosine phosphorescence intensity by a factor of 2, i.e. the probes were seen to quench at the external liposome surfaces and there remained the phosphorescence caused only by erythrosine molecules in the liposome interior. This points to the fact that free Co 2+ does not penetrate through the membrane. As C60-AD were added, the erythrosine phosphorescence decreased to zero, thus indicating the penetration of C60-AD in complex with Co 2+ through the membrane. The registration of the absorption spectra of C60-A1 and C60-A1-A1 in the presence of Co '-+ ions shows that as the concentration of Co 2+ increases, the absorption intensity is seen to grow at ~. = 270 nm suggesting the formation in the solution of complexes. So, the amino acid derivatives of fullerene C60 form complexes with bivalent metal ions Co 2+, and act as transport agents for ions through membranes into the inner volume of liposomes.

This investigation was supported by the Russian Foundation for Basic Research and by the Russian Foundation of the Scientific-technical program "Fullerene and Atomic Clusters"

300 Journal of lnorganic Biochemistry 86 (2001)

Sm(llI) complexation with biologically active ligands

Eduardo K r e m e r a Jul ia Torres a, Car los K r e m e r a, , Sixto D o m i n g u e z b, Al f redo Mede ros b, Juan M .

Arrieta c

a C6tedra de Quimica lnorg6nica, DEC, Facul tadde Quimica, CCl157, Montevideo, Uruguay. b Departamento de Quimica lnorg6nica, Universidad de La Laguna, La Laguna, Tenerife, Spain. c Departamento de Quimica Inorgdnica, Universidad del Pais Vasco- E. H. U., Bilbao, Spain

(ekremer@b ilbo. edu. u y)

Sm(III) coordination compounds are currently used as radiotherapeutic agents. It is al show enhanced intake of some biologically important molecules such as ct-aminoacids (aa) and small peptides. With this in mind, the study of samarium complexes with these ligands has obvious interest. In this work, potentiometric studies of Sm(III) in the presence of aa and small peptides have been carried out. Experiments were performed in aqueous solution ([Sm 3÷] = 6.0raM) at 37°C and 0.15M NaC104. Mono and dinuclear species were detected in the studied pH range. These complexes are not very stable, so Sm(OH)3 competes with the formation of them.

In a second stage of the present work, Sm(III) complexes with polyaminocarboxilic [ acids (edta, nta, ida) alone and with some aa were studied in order to explore the possibility to obtain more stable mixed ligand complexes of Sm(III) with aa.

Stable species, for example Sm(III)-nta-aa, were detected in solution at physiological conditions. In addition, the structure of Na3[Sm(ida)312NaC1Oa6H20 has been solved by X-Ray diffraction methods.

This work was partially supported by PEDECIBA Quimica, Uruguay;Project PM98-0148, Spain, COFI2000/11 (Gob. Canary Islands) and AECI, Spain-Uruguay (2000).

Novel bacterial molybdopterin enzymes: Mo-transhydroxylase and W-acetylene hydratase

Peter M.H. Kroneck, Dietmar J. Abt, Holger Niessen, Bernhard Schink Fachbereieh Biologie, Universitiit Konstanz, Postfach 5560 M665, 78457 Konstanz, Germany (e-mail: peter, [email protected])

Mo-dependent pyrogallol-phloroglucinol transhydroxylase (MOTH) and W-dependent acetylene novel molybdopterin containing enzymes isolated from anaerobic bacteria ~. MoTH and WAH belong to the family of DMSO reductases and catalyze non-redox reactions. MoTH is a 133.3 kDa heterodimer (100.4 and 31.3 kDa subunits). The heterodimer contains 12 Fe, 1 Mo, 12 acid-labile sulfides, and 2 molybdopterin guanine dinucleotide (MGD). The figure shows experimental and simulated EPR spectra (9.5 GHz, 14 K, 0.6 roW) of the Fe-S centers in dithionite-reduced MOTH. WAH is a monomeric enzyme with a molecular mass of 83 kDa. It carries one [4Fe-4S] cluster and one W is coordinated to MGD; selenium was absent z. In this contribution biochemical, spectroscopic, and structural aspects of the two enzymes will be presented.

hydratase (WAH) are

3200 3~00 ~eoo ~ ' 0

1. Kisker C., Schindelin H., Baas D., R6tey J., Meckenstock R. and Kroneck P.M.H., FEMS Microbiol. Rev., 22, 503- 521 (1999) 2. M e c k e n s t o c k R., K r i e g e r R, Ens ign S., K r o n e c k P .M.H. and Schink B., Eur. J. B iochem. , 264,

176-182 (1999)

The Deutsche Forschungsgemeinschaft is greatfully acknowledged for financial support.