abstracts poster presentations (z12)

Journal of Inorganic Biochemistry 86 (2001) 481

Electron transfer-independent catalysis by naphthalene dioxygenase

Mat t D. W o l f e , J o h n D. L i p s c o m b

-Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455, USA (e-mail: wolfeOOg@tc, umn. edu)

Naphthalene 1,2-dioxygenase (NDOS) is the 3 component system from Pseudomonas catalyzing the hydroxylation of naphthalene to (+)-cis-(1R,2S)-dihydroxy- 1,2-dihydronaphthalene utilizing 02 and NADH as co-substrates. Electrons are transferred from NADH through the reductase (NDR) and ferredoxin (NDF) components to the Rieske Fe-S and mononuclear iron centers of the oxygenase component (NDO). O2 activation and insertion into naphthalene are thought to occur at the mononuclear Fe site. Recently, we have shown that NDO alone is capable of a single turnover reaction when both centers are artificially reduced, l One electron oxidation of the Rieske cluster and oxidation of the mononuclear Fe(II) to Fe(III) provides the 2 electrons required for catalysis. Importantly, 02 activation requires both the reduction of the Rieske cluster and binding of naphthalene, ensuring that the 2 electrons are coupled to product formation. In this study, we have investigated the ability of NDO to catalyze oxygenase chemistry when the electron(s) and oxygen are supplied together as reduced oxygen species. Reactions of NDO with O~--H202 were analyzed and compared to the previously described NDO single turnover results. We show that, despite having oxidized Rieske cluster, NDO binds substrates and catalyzes chemistry typical of NDOS when provided with peroxide. This observation is analogous to the peroxide shunt mechanisms of cyt. P450 and methane monooxygenase. Using these enzymes as prototypes, a model is proposed for the activated oxygen species of NDO, and a mechanism for cis-dihydroxylation is discussed.

1. Wolfe, Matt D., Parales, Juan V., Gibson, David T., and Lipscomb, John D. J. Biol. Chem. 276(3), 1945-1953 (2001)

The National Institute:~ of Health grant GM-24689 is acknowledged for financial support.

Copper complexes of Tris(pyrazoly-l-yl)methane as models for copper containing nitrite reductases

Kwok-Yin Wong, Kin-Wai Yeung Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hunghom, Kowloon, Hong Kong, China (e-mail: [email protected])

X-ray crystallographic studies on several copper-containing nitrite reductases (Cu-NiRs) reveal that three histidine donors are coordinated to the pseudo-tetrahedral copper site in which nitrite binding and subsequent activation process occur [1,2]. These findings have prompted our study on water soluble copper complexes with the tridentate N-donor ligand tris(pyrazoly-1-yl)methane (tpm) as models for this class of enzymes.

The X-ray structures of some Cu(II) and Cu(I) complexes of tpm have been determined. The X-ray structure of [Cu(tpm)(NO2)(ONO)] is shown below. This complex is a catalyst for the electrochemical reduction of NO2 to NO and N20 in aqueous media as shown by the cyclic voltammograms.

~ o

°

I ~ o ~ m l i J l m v vl 5 C E

482 Journal of Inorganic Biochemistry 86 (2001)

Synthesis, structures, electrochemistry and catalytic properties of cis-dioxo-W(VI) and -Mo(VI) complexes with novel asymmetric NeOS,

and symmetric NeS2, NOSe tripodal ligands

Yee-Lok Wong, Jonathan R. Dilworth Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, UK.

(e-mail." Yee-lok. [email protected], uk)

High-valent oxo-molybdenum and -tungsten complexes have received considerable relevance to oxidation processes in industry and biological systems. Recently, we have prepared a new class of N-capped (di-/tri-valent) tripodal ligands Hx Ln (x = 2, n = 1-6; x = 3, n = 7, 8), which possess pendant arms with N, O, S donor groups and contain a wide range of substituents and with different chelate ring-size upon complexation. Treatment of these ligands with [WO2C12(DME)] (DME = 1,2-dimethoxyethane) in the presence of base (Et3N or KOH) leads to the formation ofcis-dioxo-W(VI) complexes of the types [WO2(Ln)] (n = 1-6) or K[WO2(L")] (n = 7, 8). Reaction of these ligands with [MoO2(acac)2] (acac = acetylacetonate) gives the corresponding Mo(VI) analogs. All these new complexes have been spectroscopically and structurally characterized. The electrochemistry and catalytic properties of these compounds have also been examined.

attention because of their

02

C~ CID e9

1 Johnson, M. K.; Rees, D. C.; Adams, M. W. W. Chem. Rev. 1996, 96, 2817-2840. 2. Hille, R. Chem. Rev. 1996, 96, 2757-2816. 3. Modern Coordination Chemistry: The Contributions of Joseph Chatt; Dilworth, J. R.; Arnold, P. A.; Morales, D.;

Wong, Y.-L.; Zheng, Y. (Submitted) We thank the Croucher Foundation for financial support.

Heme protein-protein interactions studied by NMR

Jonathan A.R.Worrall, Alexander Volkov, Gerard W.Canters, Marcellus Ubbink. Gorlaeus Laboratories, Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands (e-mail. j. worrall@chem, leidenuniv, nl)

Protein-protein interactions are an important feature of a variety of biological processes. In general, protein- protein complexes can range from static to transient. Static complexes are characterized by a slow dissociation rate, and the partners in the complex usually bind strongly in a single well-defined orientation. In contrast, transient complexes form when a high turnover is required such as in electron-transfer processes. In many cases the formation of electron- transfer complexes is influenced by long-range electrostatic interactions leading to the formation of an .encounter complex. This encounter complex can then sample a number of orientations of equal energy within the binding domain, with the optimal orientation for efficient electron-transfer being achieved by short-range forces such as hydrophobic contacts and H-bonds.

Advances in heteronuclear NMR in combination with paramagnetic NMR spectroscopy offer new possibilities for studying these dynamic redox protein-protein interactions. Here, we present NMR studies on two heine redox complexes: yeast cytochrome c/cytochrome c peroxidase and bovine cytochrome bflmyoglobin. Studies have revealed valuable information concerning these transient complexes, regarding 1) the type of interaction (i.e. electrostatic vs hydrophobic) 2) oxidation state dependencies for binding and 3) changes occurring away from the interface upon complex formation. For further characterization of these transient complexes the use of site-directed spin labeling has been employed and will be discussed.

This work was supported by TMR Haemworks Contract FMRX-CT98-0218.


Synthetic heme/non-heme diiron complexes: models for the active site reactivity of nitric oxide reductase (NOR)

Ian Mo Wasser," Telvin D. Ju, a Hongwei Huang, b Pierre M6enne-Loccoz, b Kenneth D. Karlin a

Department of Chemistry', Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD, 21218, USA (e-mail, [email protected])

~' Department of Biochemistry and Molecular Biology, Oregon Graduate institute, 20000 N. W. Walker Road, Beaverton, OR, 97006, USA

Nitric oxide reductase (NOR), isolated from Paracoccus denitrificans, is an enzym~ coupling of nitric oxide to nitrous oxide in bacteria. The oxidized active site is believed to contain an oxo-bridged high spin heme b with an adjacent non-heme iron] Towards modeling this heme/non-heme diiron site, we have synthesized a pair of both oxidized (Feni-O-Fe m) and reduced (Fen...Fe n) heine/non-heine diiron complexes. Our ligands employ a fluorinated-tetraphenylporphyrinate tethered, by way of an ether linkage, to a tris(2-pyridylmethyl)amine (TMPA) moiety. We present the synthesis of both the oxidized and reduced complexes. Furthermore, reactivity studies involving the reduced complex and substrates such as dioxygen and nitric oxide have been undertaken and will be discussed. The full characterization, including in-situ Fourier-transform infrared and resonance Raman spectroscopies, will also be presented in support of starting materials, proposed reactive intermediates, and end-products.

responsible for the reductive

d-- \ , ~,.,.- F e ~,...~..TT'~

i. M6erme-Loccoz P., Richter O.-M., Huang H.-w., Wasser I.M., Ghiladi R.A., Karlin K.D., de Vries S., J. Am. Chem. Soc., 122, 9344-9345 (2000)

A multitude of normal and abnormal heme peroxidases in plants

Karen G. Welinder, Laurent Duroux

Institute of Biotechnology, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Denmark (e-mail." [email protected])

Genome, transcriptome, proteome and metabolome are the key words in a new era of large-scale and systematic research in biology. High-throughput technologies have provided access to a wealth of novel gene sequences. The complete genome sequence of the model plant Arabidopsis has revealed close to 26,000 genes (Nature (Dec 2000) 408, 796-815). For almost 10 years many of the corresponding cDNA-clones (expressed sequence tag, EST) have been available for recombinant expression and structure-function studies. We have found 50 cDNA clones encoding different class III peroxidases and recently identified a total of 73 prx genes in the Arabidopsis genome. An amazing structural diversity was found, with 50% of the putative mature peroxidases showing less than 38% amino acid, and only 5% showing greater than 50% identity. Several plant peroxidases have been produced in E. coli and their crystal structures, spectroscopic and kinetic properties analyzed. (Supported by the EU BIO4 and TMR programs, and the Danish SNF and SJVF Research Councils.)


Inorganic chemistry of marine adhesives

Jonathan J. Wilker, Mary J. Sever, David L. Linson Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA

Marine organisms such as the common blue mussel (Mytilus edulis) affix themselves to surfaces by producing a protein- based glue. The soluble protein precursor contains high levels of 3,4-dihydroxyphenylalanine (DOPA) and cross-links into a hardened matrix for adhesion. Interesting properties of this biomaterial include a transition metal content 100,000 times that of the surrounding waters. We are interested in characterizing the adhesive cross-links in order to develop medical glues and improved antifouling strategies (e.g., keeping barnacles and mussels off ships). Whole protein studies determine essential reagents (e.g., metals, oxidants, enzymes) for cross-linking and optimization of material properties. Peptide mimics are used to determine the exact nature of these cross-links. We will show that soluble protein can be transformed into competent adhesives by addition of metal salts. Similar cross-linking of model peptides yields soluble products for detailed characterization.

We thank the National Science Foundation Faculty Early Career Development Program (CAREER) for support of this work~

(Nitrosyl)iron(II) porphyrinates: structural characterization and temperature- dependent mSssbauer studies of a series of five- and six-coordinate complexes.

Graeme R. A. Wyllie a, Charles E. Schulz b, W. Robert Scheidt a Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN

46556-5670, USA. (e-mail wyllie, [email protected]) b Department of Physics, Knox College, Galesburg, IL 61401, USA.

We report new studies of five- and six-coordinate (nitrosyl)iron(II) porphyrinates. Structural characterization of previous six-coordinate iron(II) systems with neutral N-donors have shown that the nitrosyl ligand exerts a strong trans effect making such complexes difficult to isolate. We report new six-coordinate derivatives and their characterization. The trans effect is supported by the observed lengthening of the axial bond trans to the nitrosyl in these new complexes and spectroscopic evidence is provided for these complexes. Temperature-dependent M6ssbauer spectra have been measured for a series of five-coordinate (nitrosyl) porphyrmates. Asymmetric quadrupole doublets are observed for all tetraaryl porphyrinates while the octaethylporphyrin complex shows a single symmetric quadrupole doublet. The X-ray structure of this latter complex exhibits a single well-def'med orientation of the nitrosyl ligand, whilst structures of the tetraaryl systems show substantial disorder of the nitrosyl, a fact thought to be reflected by the M6ssbauer spectra. Additionally an unusually rapid drop in the absorption area with temperature is observed, corresponding to a Debye-M6ssbauer temperature of 140 K for OEP and 80-100 K for the tetraaryl systems.

We thank the US NIH (GM-38401) for support.


Nitrite adduct to Cu(I) and Cu(II) complexes containing tridentate ligand with aromatic amines as models for the active site of copper nitrite reductase

Kazuya YamaguclLi, Hiroshi Yokoyama, and Shinnichiro Suzuki Department of Chemistry, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043, JAPAN(e-mail: [email protected])

Nitrite reductase (NIR), a key enzyme of denitrification, catalyzes the reduction of nitrite to nitrogen monooxide. 1 Copper-containing NIR contains each of type 1 Cu (blue copper) and type 2 Cu (nonblue copper) per a monomer. The type 2 Cu site is bound by three His residues and one solvent water, and results in a distored tetrahedral geometry. We report herein the structural and functional characterization of copper(I) and copper(II) complexes with nitrite and a tridentate ligand containing aromatic amines as models for type 2 Cu site of Cu- NIR. X-ray crystal structures of [Cu(Me2bpa)(NO2)(CIO,)] (1) and [Cu(Me2bpa)(NO2)]2"[(Ph3P)2NPF6] (2) are depicted m Figure. The structure of 1 has the asymrnetric~O,O-nitrite

[Cu2÷(Mo2bpa)(NO2)(CIO,)] [Gul*(Mo2bpa)(NO2)]

chelation to the octahedral Cu(II) ion with a Mezbpa and a perchlorate ligands, while 2 is coordinated with a nitrite through nitrogen and has a distorted tetrahedral geometry. When 4 equiv, of glacial acetic acid was added to a solution of 2 in CH2C12 at room temperature, the color of the solution was changed from yellow to green and the evolution of NO was observed by gas chromatography and ESR spectroscopy using a trapping agent of NO, Fe(DTCS)2.

1. Suzuki, S., Kataoka, K., and Yamaguchi, K., Acc. Chem. Res., 33,728-735 (2000).

This work was supported by Grant-m-aid for Scientific research from MEXT of Japan, to whom we express our thanks.

Thermal stability of copper-dioxygen complexes with substituted tripodal pyridylamine ligands having hydrogen bonding site

Syuhei Yamaguchi, Akira Wada, Koichiro Jitsukawa, Hideki Masuda Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan

Copper ion plays an important role for capturing and activation of dioxygen in biological and catalytic processes. Coordination modes of peroxo dianion for dicopper(II) are known to depend on the stereochemistry of the coordination environment. Copper(I) complexes with tetradentate tripodal ligands such as tris(2-pyridylmethyl)amine (TPA) and bis(2-pyridylmethyl)(6-methyl-2-pyridylmethyl)amine (6-MeTPA) (Figure 1) have been reported to form trans -1,2-peroxo dicopper complexes. We investigated thermal stability of the copper-dioxygen complexes with tetradentate tripodal ligands having hydrogen bonding site, such as bis(2-pyridylmethyl)(6- amino-2-pyridylmethyl)amine (MAPA) and bis(2-pyridylmethyl)(6-pivalamido-2- pyridylmethyl) amine (MPPA) (Figure 1) that form similar peroxo species. The stability of Cu(MAPA)-Oz complex is superior to that of Cu(6-MeTPA)-O2 complex, although they have both bulky substituent groups at 6-position. The above finding indicates that the hydrogen bonding amino group for MAPA contributes to the stabilization of trans -1,2-peroxo species.

~ R

= C H 3 : 6 - M e T P A

= N H z : M A P A

= N H C O t B u : M P P A

Figure 1. Tripodal ligands used


UV Resonance Raman spectroscopic studies of redox state dependent behavior of histidine residues in pseudoazurin

Sachiko Yanagisawa a, Michihiko Aki b, Teizo Kitagawa b, Takamitsu Kohzuma a Faculty of Science, lbaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan

(e-mail [email protected], ibaraki.ac.jp) h Institute for Molecular Science, Myodaiji, Okazaki, Aichi 444-8585, Japan

Pseudoazurin is a blue copper protein revolved in the nitrite respiration electron-transfer system of several denitrifying bacteria. Histidine residues of pseudoazurin are known to show unique behavior dependent on the redox state and pI-I conditions. Oxidized blue copper proteins are well known to show characteristic spectroscopic properties in the UV-Vis, resonance Raman, and EPR spectra, however, the reduced blue copper proteins normally show featureless spectroscopic properties because of d ~° character. Recent development of UVRR method enable to obtain structural information from such featureless reduced blue copper center.

UVRR spectra of oxidized pseudoazurin provide characteristic Raman bands from histidine imidazole moiety in D20 solution at various pH values (Figure 1). The observed Raman bands at 1385, 1395 and 1405cm ~ are assigned to H81, H40 and H6 imidazoles, respectively. UVRR spectra of reduced pseudoazurin indicates the lower frequency shift of H81 and H40 imidazole Raman bands probably due to 7~-back donation from Cu ~+ to coordinated histidine imidazole groups.

pH*7.6

pH*6 9

pH*5.7

pH*4 9

1395 (H40)

1405 (H6)

1385 (H81~

1550 1500 1450 1400 1350 1300 Raman shift / cm -1

Figure 1 UVRR spectra (ex. 244 nm) of oxidized pseudoazurin at various pH*.

Unprecedented sugar-dependent in vivo antitumor activity of carbohydrate- pendant cis-diamminedichloroplatinum(II) complexes

Shigenobu Yano a, Yoshie Shinohara a, Izabela Brudzifiska a, Yuji Mikata b, and Chris Orvig a

"Division of Material Science. Nara Women's University, 630-8506, Kitauoya-Higashi-machi, Nara, Japan, (e-mail yano@cc, nara-wu, ac.jp) bDepartment of Chemistry, Nara Women "s University, 630-8506, Kitauoya-Higashi-maehi, Nara, Japan CMedicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, Vancouver BC, V6T 1Z1, Canada

We report a systematic synthesis of carbohydrate-pendant cisplatin derivatives and their in vivo antitumor activity. Eight carbohydrate-pendant platinum(II) complexes have been synthesized (4-55% yield). D-Glucose, D-mannose, D- galactose, D-xylose, and L-glucose are attached to the dichloroplatinum (II) moiety by 1,3- or 1,2-diaminopropane chelates through with an O-glycoside bond. This series of compounds includes the c~ and 13 anomers for the D-galactose derivative (3 and 4), the D and L isomer for the glucose derivative with a chiral 1,2-diaminopropane linker (6 and 7), and both six and five membered chelate ring size isomers with D-glucose (1 and 6) and D-xylose (5 and 8).

The anticancer activity of these platinum (II) complexes was investigated against P388 cells implanted in mice. Cancer cells (106 of P388 cell) were transplanted intraperitoneally (ip) into CDF1 mice (6 mice/group), followed by ip administration of the drugs to the mice on days 1 and 5. The mean survival time of the treated group (T) was compared with that of the untreated control group (C). A T/C value of 194% was obtained for the Ct-D-galactose derivative 3 at 400 mg/kg dose, whereas the [3-D-galactose derivative 4 showed low toxicity at the same dose. Significant toxicity was observed for the mannose derivative 2, even at a dose of 100 mg/kg. Because the LD50 of cisplatin is approximately 10 mg/kg, all the carbohydrate moieties reduced the toxicity inherent with platinum (II) complexes. The linker structure also affects the antitumor activity of 1 versus 6/7, or 5 versus 8; however, D- and L-glucose derivatives (6,7) showed similar dose/activity profiles.

Journal of lnorganic Biochemistry 86 (2001) 487

Structure and function relationships in bacterial hemoglobins

_Syun-Ru Y e h a, M a s a h i r o M u k a i a, P i e r r e - Y v e s S a v a r d b, Y a n n i c k Ouel le t b, M i c h e l Guer t in b, Ca ther ine

E. Mi l l s c, R o b e r t K. P o o l e c,

adept, of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY I0461,USA (email: [email protected])

bDept, of Biochemistry, Laval University, Quebec, G1K 7P4, Canada CDept. of Molecular Biology & Bioteehnology, The University of

Sheffield, Sheffield S10 2 TN, UK .~ 7

Recent discoveries of hemoglobins in several bacteria and fungi introduce a new ~ 6 twist to the common perception that hemoglobin is an oxygen carrier, in view of the o 5 fact that oxygen delivery is a diffusion-controlled process in these unicellular ~ 4 organisms, in this work, the structure-function relationships in three bacterial N 3 hemoglobins, two from M. tuberculosis (HbN and HbO) and one from E. coli ~

~uD 2 (Hmp), were studied. The physiological functions of HbN and HbO are not o established yet. Hmp, on the other hand, has been demonstrated to be a dioxygenase 1 that converts NO to nitrate. Preliminary studies performed with resonance Raman 0 spectroscopy indicate that the active site structure of Hmp is analogous to peroxidases rather than vertebrate globins, indicating that the heine pockets of this bacterial hemoglobin may be tailored to perform NO/oxygen chemistry. Similar structural features and possible functions were demonstrated in HbN, but not relationships in these bacterial hemoglobins will be discussed.

1 2 3 4 5 6 7

log k calculated 12

in HbO. The structure / function

t. ~'Flavohemoglobin (Hmp): A Globin with A Peroxidase-Like Catalytic Site" Masahiro Mukai, Catherine E. Mills, Robert K. Poole and Syun-Ru Yeh, J. Biol. Chem., 276, 7272-7277, 2001. 2~ "A cooperative oxygen binding hemoglobin (HbN) from Mycobacterium tuberculosis: stabilization of heme ligands by a distal tyrosine residue" Syun-Ru Yeh, Manon Couture, Yannick Ouellet, Michel Guertin & Denis L. Rousseau, J. Biol. Chem., 275, 1679-1684, 2000.

Applicat ion of the Marcus Cross Relation to Hydrogen Atom Transfer Reactions of Transit ion Metal Complexes

Jeffrey C. Yoder, a Justine P. Roth, ~ Tae-Jin Won, b James M. Mayer ~ Department of Chemistry, University of Washington, Campus Mail 351700, Seattle, WA 98105- 1700 USA (e-mail: [email protected])

b Department of Chemistry, Changwon National University, 9, Sarim, Changwon, 641-773, Korea (ROK)

Hydrogen atom abstraction reactions are important components of the reaction cycles of enzymes as well as industrial oxidations and aerobic autoxidation. We have developed iron and cobalt coordination compounds which can oxidize organic compounds with weak X-H bonds (X = C, O) by a hydrogen atom abstraction mechanism. Hydrogen atom self exchange rates have been measured for these transition metal species, with kH. for the iron derivatives on the order of 10 4 M ~s -~. Hydrogen atom transfer reactions were carried out between these transition metal species and hydroxylic organic compounds for which hydrogen atom self exchange rate constants are also known. The Marcus cross relation was then utilized to predict rate constants for these reactions with good agreement to observed values. Implications of these results for the understanding of the mechanism of hydrogen atom transfer will be discussed.


A theoretical study of the dynamical aspects of alkane hydroxylation by compound I of cytochrome p450

Kazunari Yoshizawa, Takashi Kamachi, Yoshihito Shiota Institute for Fundamental Research of Organic Chemistry, Kyushu University, Fukuoka 812-8581, Japan (e-mail.'kazunari@ms. ifoe.kyushu-u, acjp)

Dynamical aspects of alkane hydroxylation mediated by compound I of cytochrome P450 are discussed from classical trajectory calculations at the B3L YP level of density functional theory. 1 The nuclei of the reacting system are propagated from a transition state to a reactant or product direction ac cording to classical dynamics on a Born- Oppenheimer potential energy surface . Geometrical and energetical changes in both low-spin doublet and high-sp in quartet states are followed along the ethane to ethanol reaction pathway, which is partitioned into two chemical steps: the first is the H atom abstraction from ethane by the iron-oxo species of compound I and the second is the rebound step in which the resultant iron-hydroxo complex and the ethyl radical intermediate make reaction to form the ethanol complex. Molecular vibrations of the C-H bond being dissociated and the O-H bond being formed are significantly activated before and after the transition state, respectively, in the H atom abstraction. The rebound process begins with the iron-hydroxo complex and the ethyl radical intermediate and ends with the formation of the ethanol complex, the essential process in this reaction being the format ion of the C-O bond. The H-O-Fe-C dihedral angle corresponds to the principal reaction coordinate for the rebound step. When sufficient kinetic energy is supplied to this rotational mode, the rebound process should efficiently take place. Trajectory calculations suggest that about 200 fs is required for the rebound process under specific initial conditions, in which small kinetic energy of 0.1 kcal/mol is supplied to the transition state exactly a long the reaction coordinate. A large part of the kinetic energy is distributed to the C-H and O-H stretching modes before and after the transition state for the H atom abstraction, respectively, and a small part of the kinetic energy is distributed to the Fe-O and Fe-S stretching modes and some characteristic modes of the porphyrm ring. The porphyrm marker modes that explicitly involve Fe-N stretching motion are effectively enhanced in the hydroxylation reaction.

1. Yoshizawa K., Kamachi T. and Shiota Y., J. Am. Chem. Soc., 123, in press (2001).

Antimicrobial and genotoxic activity of organotin(IV) complexes with butyl- and pentylisatin bisthiocarbohydrazones

Franca Zani a, Alessia Bacchi b, Mauro Carcelli b, Andrea Fochi b, Giancarlo Pelizzi b "Pharmaceutical Department, University of Parma, Parco Area delle Scienze 27/,4, 43100 Parma,

ITALY (e-mail: franca.zani@unipr, it) bDepartment o f General and Inorganic Chemistry, Analytical Chemistry, Physical Chemistry.

University o f Parma, Parco Area delle Scienze 1 7/,4, 43100 Parma, ITALY

The isatin molecule has been N-alkylated (R = n-C4H9, n-CsHll) and reacted with thiocarbohydrazide, in a 2:1 stoichiometric ratio; the bisthiocarbohydrazone ligands were able to coordinate diorganotin(IV) derivatives carrying methyl, ethyl, butyl or phenyl moieties. The ~ ~ ~.~ ~ ~ , H spectroscopic characterization of the complexes has been completed by the X-ray ~ " r-- "N . . 0 structure of some of them. The ligands and the complexes were studied by means of the ~ N - - ~ o ~ I S ~ N - - R two-fold dilution method for their in vitro antimicrobial properties against several microorganisms, such as Gram positive (Bacillus subtilis, Staphylococcus aureus) and Gram negative (Escherichia coli) bacteria, yeasts (Candida tropicalis, Saccharomyces cerevisiae) and moulds (,4spergillus niger). None of the ligands showed any antimicrobial potency (MIC > 100 gg/ml). On the contrary, the butyltin complexes were moderately effective against bacteria exhibiting MIC values ranging from 25 to 100 gg/ml. B. subtilis and S. aureus were more sensitive than E. coli. All the ligands and complexes were inactive against the tested fungi. The obtained results suggest that the presence of more than one lipophilic function doesn't enhance the antimicrobial activities of these products. The investigation of genotoxicity was performed by means of the Salmonella-microsome test. None of the synthesized compounds was recognized as a genotoxic chemical.

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

Journal of lnorganic Biochemistry 86 (2001) 489

Platinum and palladium aryl oxime metallacycles highly effective promote the P-S bond clevage in organophosphate neurotoxins

Anna I. Z a t s m a n , G r e g o r y M. K a z a n k o v , Vic tor ia S. Sergeeva , and A lexande r D. R y a b o v

Chemical Enzymology Department, Faculty of Chemistry, Moscow State University, 119899, Lenin's Hills, Moscow, Russia, ( e. mail: zatsman@enzyme, chem. msu. ru)

The hydrolysis of organophosphate neurotoxins, such as demeton-S ((EtO)2P(=S)SCH2CH2SEt, I) and malathion ((MeO)2P(=S)SCH(COOEt)(CH2COOEt), II) is powerfully promoted by the cyclometallated aryl oximes cis(C,S)- [MH(C6H3-2-CMe=NOH-5-R)CI(DMSO)] (M = Pt and Pd, R = H, F, C1, Me, and MeO, III). In the presence of the complexes, the rate of hydrolysis of the single P-S bond is increased 105-106 times compared to the rate of the base hydrolysis. Previous work L has indicated that the hydrolysis of thiophosphoric acid esters (parathion, coumaphos) proceeds catalytically in the presence of III. The results of the present study of the hydrolysis of thiol esters indicate that complexes III behave as promoters rather than catalysts in hydrolysis of I and II. The second-order rate constant for the hydrolysis of I and II in the presence of III equal 25 and 1?10 2 M -~ s -~ (25 °C, pH 9.0), respectively. The mechanism of the promoted by III ihydrolysis of I involves the substrate coordination with metal ion via sulfur atom followed by intramolecular nucleophilic attack by coordinated oximate-ion. The cleavage of I liberates the potentially good ligand HSCH2CH2SC2Hs, which on interaction with the Pt H C,N-metallacycle blocks the substrate binding site, making intramolecular nucleophilic attack impossible. Kinetic studies of the promoted by III hydrolysis of malathion suggests different mechanism compared to the that of hydrolysis of I. The results obtained are in accord with a three-step mechanism the nature of which is discussed in detail.

!. G. M. Kazankov, V. S. Sergeeva, E. N. Efremenko, L. Alexandrova, S. D. Varfolomeev, A. D. Ryabov Angew. Chem., int. Ed. Engl., 2000, 39, 3117-3119.

A general approach towards metal complex-PNA conjugates

Felix Zelder a, Ro land Kr~imer a

Anorganisch-Chemisches Institut, Ruprecht-Karls-Universiti~t Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, GERMANY (e-mail: [email protected], uni-heidelberg.de)

Nonenzymatic hydrolysis of the phosphodiester backbone of nucleic acids is an attractive research aim in molecular biology, but the sequence specific hydrolysis of ssDNA was achieved only in one case with a conjugate consisting a Ce(IV)-complex attached to a DNA-oligonucleotide. ~ The nonenzymatic sequence specific hydrolysis of dsDNA is not reported. Since peptide nucleic acids show interesting binding properties we are focussing the design of metal-complex- PNAs in a way allowing the generation of their combinatorial libraries. Their synthesis and characterization will be discussed.

1. Komiyama M., J. Biochem.; 118, 665 (1995).


Recognition of bismuth by lactoferrin and urease

Li Zh.ang, Weihong Du, Ka Yee Szeto and Hongzhe Sun* Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong

Bismuth compounds have been used for treating gastrointestinal disorders for several decades, III but the mechanism of action, particularly bismuth inhibition of the Gram-negative bacterium Helicobacter pylori (H. pylori), still remains poorly understood. The antimicrobial activities of Bi m complexes to gram-negative bacteria were previously found to be dependent on the iron uptake system, C21 and H. pylori probably acquires iron via lactoferrin (hLF) and iactoferrin receptor systems. Our electronic absorption and NMR data have shown that Bi m binds to human lactoferrin at the specific Fem sites along with either carbonate or oxalate as the synergistic anions. Bi2-hLF blocked uptake of radiolabeled 59Fe-hLF into rat IEC-6 cells, and surprisingly apo-hLF (but not apo-transferrin) was almost as effective as bismuth-loaded lactoferrin. These results indicated that both apo- and Bim-hLF could be recognized by lactoferrm receptor and taken up into cell.

Several studies have demonstrated the importance of H. pylori-produced urease for survival of the bacterium in the acidic environment of stomach. I3J Urease would therefore be an excellent target for therapy. The inhibitory effects of bismuth complexes, such as ranitidine bismuth citrate (RBC), Bi(cys)3 and Bi(GSH)3, have been inverstigated, and the possible mechanism of inhibitory effect will be discussed.

1. Banci L., Bertini I., Ferroni, F. and Rosato, A., Eur. J. Biochem., 249, 270-279 (1997) 2. P. Domenico, J. Reich, W. et al. J. Antimicro. Chemother. 1996, 38, 1031-1040 3. H. L. Mobley, L. T. Hu and P. A. Foxal, Scand. J. Gastroenterol. 1991, 187, 39-46

Investigations into the conformational changes of CuZnSOD through protein design

Soshanna G. Zittin a, Aram M Nersissian a, Hyeyeong Yeom b, Yi Lu c, and Joan S. Valentine a Department of Chemistry and Biochemistry, University o f California, Los Angeles, 607 Charles E. Young Drive East Box 951569, Los Angeles, 90095-1569, USA (email: [email protected]) Deceased

CDepartment of Chemistry, University of Illinois, Urbana, Illinois, 61801, USA

Metal binding has an enormous effect on the physical and chemical properties of copper-zinc superoxidedismutase (CuZnSOD) and could be responsible for the aberrant chemistry postulated as a cause for CuZnSOD related amyotrophic lateral sclerosis (ALS). Therefore, understanding all aspects of CuZnSOD metallation is critical. We have replaced one of the histidines in the zinc binding site with a cysteine, creating a new copper binding site ~. Two cysteine-to-Cu(II) charge transfer transistions at 450 and 600 nm in the mutant spectra provide a conformation sensitive visible probe of the zinc site environment. Upon rapid addition of Cu(II) to the apo mutant, a new feature at 410 nm is observed and slowly shifts to 450 nm indicating a change in the metal site geometry. Kinetic analysis shows a two step process with enthalpy of activation energies of 8.81 and 26.74 kcal/mol. The slower step is similar to the energy of proline isomerization. CuZnSOD has three conserved prolines near the zinc site whose isomerization could be triggered by metal binding. This conformational change may have implications in ALS as well as metal-cofactor insertion into apo-SOD.

1. Yi Lu, et al., J. Am. Chem. Soc., 115, 5907-5918 (1993)


Effect of various metals on the stability and catalytic activity of chromobacterium violaceum p henylalanine hydroxylase

J e r o m e Z o i d a k i s a, K i m V u a, M a h d i A b u - O m a r a

" Department of CJ~emistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Avenue, 90095, Los Angeles, California, U.S.A.(email: [email protected])

Phenylalanine hydroxylase from Chromobacterium violaceum catalyzes the conversion of Phenylalanine to Tyrosine. The enzyme is a monc, mer of 33.5 kDa and contains one non-heine iron(II) ~. The effect of temperature and various metals on the stability and catalytic activity of the enzyme was determined by steady-state kinetics, Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC). The half-life of the enzyme at 47 °C is 66+_4 rain in the presence of Iron(II) and 10_+ t rain in the presence of EDTA. The melting temperature of the protein determined by CD and DSC is 53+2 °C in the presence of EDTA and 63_+2 °C in the presence of Iron(II) and Cobalt(It). Iron(II) is required for catalytic activity and other metals inhibit the enzyme with Cobalt(II) and Zinc(II) having the most marked effect, The visible spectrum of the enzyme m the presence of Cobalt(II) indicates that the metal binds in an octahedral configuration at the active site. The kcat and KM for Phenylalanine were determined between 7 and 40 °C. The KM remained constant between 20 and 40 °C but rapidly increased below 20 °C. In conclusion Iron(II) is required for catalytic activity and stability and the enzyme is adapted for optimal Phenylalanine binding at temperatures above 20 °C.

1. Chen D. and Frey P.A.., Journal of Biological Chemistry, 273, 25594-25601 (1998)

The Beckman Foudation is acknowledged for financial support.

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