- Email: [email protected]
Abstracts Poster Presentations (Z9)
Journal of Inorganic Biochemisto., 86 (2001)
451
Copper complexes of 1,2,4-triazolines derivatives of c~-amino acids M. Tegoni a, M. Cingi Biagini a, E. Ferloni ~, M. Lanfranchi a, M.A. Pellinghelli a, "Dipartimento di Chimica Generale ed Inorganica, University o f Parma, Parco Area delle Scienze, 17A, 43100 Parma, I T A L Y (e-mail: [email protected], unipr, it) The study of model systems of metalloenzymes containing copper(II) is of great importance in order to understand the mechanisms of action of these proteins. Here we present the synthesis and the characterization both in solid and in solution state of new triazoline derivatives functionalized with amino acid residues and their copper(II) complexes. Ligands 1 and 2 have been synthesized by double condensation of thiocarbonohydrazide with BOC-protected L-amino acids activated on the carboxylic group followed by H N--N deprotection of the amino group. Both ligands 1 and 2 have been characterized in solution / \\ .NHa by potentiometric studies. In the case of ligand 1 the determined pKa were 6.48(1) and S 8.03(1). Copper (II) complexes with ligand 1 have been isolated and crystallized both in the N I 2 R ratio of M:L 1:1 and 1:2 and their structure determined. In all the complexes the ligand NH showed to be protonated on the amino group and coordinated to the metal ion via hydrazinic nitrogen and sulfur atoms. The complex [CuC12(1)]CI presents a square planar coordinated Ligand 1: R = H copper ion, whereas in [Cu(OH2)2(1)z](SO4)2 the copper atom is octahedrally coordinated Ligand 2: R = CH3 with two water molecules in axial position. EPR and MAGSUS experiments on the complex [CuCI2(1)]C1 in the solid state revealed an anti ferromagnetic behaviour with an inversion temperature of 19(1) K. This behaviour is most probably due to weak interactions between copper atoms and sulfur atoms of adjacent molecules, forming polymeric Cu - S - Cu chains. +
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Synthesis and characterization in solution of hydroxamic acid derivatives as new potential enzyme inhibitors M. Tegoni ", F. Dallavalle", M.A. Santos b "Dipartimento di Chimica Generale ed lnorganica, Chimica Analitica, Chimica Fisica, University of Parma, Parco Area delle Scienze, 17A, 43100 Parma, Italy. ~'Centro de Quimica Estrutural, Complexo I, Instituto Superior Tkcnico, 1049-001 Lisboa-codex, Portugal. (e-mail: masantos@popsrv, ist. utl.pO The development of suitable amino acids or short peptides containing hydroxamate groups are of current interest because they can act as inhibitors for a large number of metalloenzymes. Herein we present two new potential metalloenzyme inhibitors L1 and L2 (H2L). They 0 Ph incorporate one hydroxamate and one monopeptide-carboxylate group at terminal L1 HO. fl]~ / ~ .~L ~J positions for metal binding and enzyme-inhibitor interaction, respectively. Besides ~- v -,f - , , f o COOH the synthesis of the compounds, we report the equilibrium studies with Cu 2+, Ni 2+ and Zn 2+ The preparation of the ligands required standard protecting-group o techniques and it mainly involved the connection of the two molecular units, L2 H O ' - ~ / I vI ~ / ~ ]~- S - .,() through a peptide bond, followed by formation of the hydroxamic acid. Complex formation studies with this set of metal ions revealed the existence of the o COOH corresponding ML, ML2 and ML2H_t species. In the case of Ni z+, the ML3 species was also found. All the calculated constants are very close to those determined for monohydroxamic acids. In the case of copper(lI), the solution containing CuL2 and CuLzH.I are green 0q~,~ ca. 630 run), showing that the coordination mode is a 2 x {0,0} chelation on the hyrdoxamate group. The species CuL2 has most probably a planar coordination geometry, whereas in ZnL2 the metal is probably tetrahedral. ML2H.j species are most probably deprotonated on one hydroxamate nitrogen atom, rather than forming an hydroxo specie with an OH- coordinated in an axial position. The authors thank to the COST D21 program for financial support.
452
Journal of lnorganic Biochemistry 86 (2001)
The interaction of the substrate analoguep-nitrophenol with the oxidised type-3 copper protein tyrosinase studied by paramagnetic NMR A . W . J . W . T e p p e r a, L . B u b a c c o b, M . U b b i n k a, U . K o l c z a k a G . W . C a n t e r s a
'%eiden institute of Chemistry, Leiden University, Einsteinweg 55, 2300RA Leiden, The Netherlands bDepartment of Biology, University of Padua, Via Trieste 75, 30121 Padua, Italy We have recently shown that the oxidised [Cu(II)-R-Cu(II)] form of the type-3 copper protein tyrosinase (Tymet) from Streptomyces antibioticus is amenable to study by paramagnetic NMR methods, providing a sensitive probe of the active site coordination [1]. The binding of exogenously added inhibitors leads to drastic changes in the paramagnetic part of the 1H NMR spectra. Here we report on a paramagnetic NMR study of the interaction of the monodendate suhstrate analogue p-nitrophenol with native Tymet and its halide bound derivatives. A titration of Tymet with p-nitrophenol showed that the electronic active site smacture remains largely unperturbed upon binding of the ligand. Titrations of the F- and C1- bridged Cu2 derivatives of Tymet with p-nitrophenol induced substantial changes in the paramagnetic ~H spectra in terms of Hisproton signal positions and linewidths. The ligand appeared to be in fast exchange with either TymetF or TymetC1. In both cases, three sharp HisN a proton signals appeared more sensitive to the presence ofp-nitrophenol than the other remaining three, possibly indicating that the monophenol interacts more strongly with one copper ion in the dinuclear site. The nitrophenol / Tymet exchange process appeared to induce significant broadening of the 2 diamagnetic p-nitrophenol ring proton signals in the bulk. Determination of both T~ and T2 relaxation enhancements of the bulk nitrophenol signals in samples containing various [nitrophenol]/[Tym~t] allowed estimation of the ligand dissociation constant Kd and the longitudinal and transversal paramagnetic relaxation rates of Tymet bound nitrophenol, showing that the monophenol is directly coordinated to copper through the hydroxyl moiety. The relevancy of the findings for understanding the catalytic mechanism is discussed. 1. Bubacco, L., Salgado, J., Tepper, A.W.J.W., Vijgenboom, E., Canters, G.W., FEBS Letters 442, 215-220, 1999.
A novel class of pH-sensitive paramagnetic CEST contrast agents for MRI applications Enzo Terrenoa,SilvioAime a, Alessandro Bargea, Daniela Delli Castellia, Flemming Ulrich Nielsenb ~' Dipartimento di Chimica 1FM, Universit~ di Torino, Via P. Giuria 7, I-10125, Torino, Italy Laboratorio lntegrato di Metodologie Avanzate, Bioindustry Park Canavese, Via Ribes 5, 110010, Colleretto Giacosa (TO),Italy, (e-mail: [email protected]) The recently introduced new class of Contrast Agents based on Chemical Exchange Saturation Transfer (CEST) may have a huge potential for the development of novel applications in the field of MRI. Many diamagnetic small organic molecules (aminoacids, sugars, heterocyclic compounds) as well as macromolecular systems (dendrimers and polymers) have been so far investigated. 1 A good CA for CEST application has to possess mobile protons whose exchange rate with water is as high as possible before coalescence takes place. This means that larger shift separation between the two exchanging ~ ~.N ~.;~'/"<° ~ sites results in an enhanced CEST effect. Recently, Sherry et al. showed that a particularly o:j[,..~ useful source of highly shifted exchangeable protons can be provided by the slowly exchanging .~ %j~o~ water protons bound to a paramagnetic Eu(III)-chelate. 2 An alternative source may be represented by labile protons on the ligand of paramagnetic complexes. In this contribution, we present the properties of a series of Ln(III)-chelates of a tetraglycineamide derivative of DOTA, which contains four equivalent labile amidic protons in close proximity of the paramagnetic center (see figure). The best CEST effect has been obtained for the Yb(III) derivative. Interestingly, the effect is markedly pH-dependent. A ratiometric method for the "in vivo" pH measurement has been developed which allows to observe a CEST effect not longer dependent on the absolute concentration of the CA. 1. Ward K.M., Aletras A.H. and Balaban R.S., J. Magn. Res., 143, 79-87, 2000. 2. Zhang S., Winter P., Wu K., Sherry A.D., J. Am. Chem. Soc., 123, 1517-1518, 2001.
Journal of lnorganic Biochemistry 86 (2001)
453
NMR detection of the inner-rotatiopn of water around its coordination axis in Eu(III) complexes Enzo Terreno a, Silvio Aime, Alessandro Barge, Mauro Botta, Erik Bruno Dipartimento di Chimica IFM, Universitd di Torino, Via P. Giuria 7,1-10125, Torino, Italy The huge amount of work carried out in the last decade in the field of Lanthanide(llI) complexes has provided an in-depth understanding of their solution structures and dynamics. In particular it has been possible to get a good understanding of the main determinants of the exchange rates (kex) of the coordinated water molecule. On the basis of ~70-NMR investigations on a large number of coordination schemes, it has been found that the exchange lifetime rM (1/kex) c o v e r s almost 6 order of magnitude (fi'om l0 -9 to 1 0 -3 S!). 120 ~o.~ The cationic tetrabenzylglycinamide-DOTA Eu(lll) complex (Eu-DOTAM-GIyBz) owns ,0~ . ~ , ~ ~ , one of the longest zTv.,(0.59 ms at 298K). Thus, on this system, it has been possible to ~0measure the relaxation time T ~ of the resonance of the coordinated water over an~ ~01 extended range of temperature (solvent CD3CN) and to compare the observed behaviour ~ ~ ! with that of 1H-resonances of the macrocyclic ligand. The analysis of the obtained I/Tt ~,,i "'!'" ° : " i vs temperature profiles allows to assess that the inner rotation of the coordinated water ~ I 0[ . . . . . --occurs on a faster timescale than the overall reorientation of the complex. Analogous ~ _0 _,~, ~0 ~0 3o0 ~0 ~ 360 results were obtained on related Eu(llI) complexes. J- K On the basis of the close analogy between Eu(III) and Gd(lll) complexes, this finding suggests that a new item has to be considered in the design of contrast agents endowed with enhanced relaxivities. .
Electron tunneling in cytochrome c crystals F. Akif Tezcark Brian R. Crane, Jay R. Winkler, Harry B. Gray Beckman Institute, California Institute of Technology, Pasadena, CA 91125, U.S.A. The factors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the very short time that tunneling occurs. In order to circumvent this structural ambiguity, we have measured and analyzed the kinetics of electron 1013 Electron Tunneling transfer (ET) b e t w e ~ native and Zn-substituted tuna cytochrome c (cyt c) ps Timetable molecules in crystals of known structure (1.5 ~. resolution). ET rates (320 s 1011 10 lo for *Zn-cyt c --+ Fe(lll)-cyt c; 2000 s-' for Fe(II)-cyt c --~ Zn-cyt c +) over a ZnFe distance of 24.1 A closely match those for intraprotein electron tunneling nS ~> protein over similar donor-acceptor separations. These results indicate that van der 10 -7 ~[3 [3 . time Ms ,/~a v ~ o o Waals interactions and water-mediated hydrogen bonds are effective coupling elements for tunneling across a protein-protein interface. Zn-porphyrin lO ~ eg photoreactions should be particularly useful for the study of ET in other heme ms lO ~ protein crystals, as well as for the generation of short-lived redox intermediates io-i that are amenable to structural characterization. S vac~Jum H~O | ,
This project was supported by the Arnold and Mabel Beckman Foundation, the National Science Foundation and the Helen Hay Whitney Foundation.
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Journal of inorganic Biochemistry 86 (2001)
454
Kinetic and equilibria studies of the aquation of the trinuclear platinum anticancer agent BBR3464 Donald S. Thomasa, Murray S. Davies a, Alexander Hegrnansb, Susan J. Berners-Pricea, Nicholas Farrellb "Department of Chemistry, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009 AUSTRALIA. (e-mail. [email protected], edu.au) ~Department of Chemistry, Virginia Commonwealth University, 1001 Main Street, Richmond, Virginia, 23284-2006, USA. BBR3464, which breaks the old barriers of activity predicated by cisplatin, is providing a fresh perspective in the field of platinum anti-cancer drugs. [1H,15N] HSQC NMR spectroscopy has been used to investigate the hydrolysis profile of BBR3464 under conditions of different ionic strength and temperature and in the presence of phosphate. Rate and equilibrium constants for the aquation of BBR3464 have been determined, together with the acid dissociation constants for the aquated species. The equilibrium lies further towards the aquated species than occurs for the related dinuclear complex 1,1/t,t (n=6) ~ under the same conditions, while the pKa value(s) of the aquated drug is identical (5.62). The presence of phosphate does not slow the initial aquation of BBR3464 but reversible reaction between aquated species and phosphate does occur.
1. Davies, M. S., Cox, J. W., Bemers-Price, S. J., Barklage, W., Qu, Y and Farrell, N., Inorg. Chem., 39, 1710-1715 (2000). The authors thank the Australian Research Council, U.S. National Institute of Health, U.S. National Science Foundation and the American Cancer Society for financial support.
Stopped-flow time-resolved infra-red spectroscopy of the activation of small molecules by nitrogenase John D. Tolland, Gillian A. Ashby, Simon J. George, Roger N.F. Thorneley Biological Chemistry Dept. John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH (e-mail: [email protected]) We have been using stopped-flow Fourier transform infra-red spectroscopy (SF-FTIR) to monitor the dynamics of inhibition of nitrogenase by carbon monoxide. SF-FTIR gives the first real-time measurement of consumption of a substrate, azide, through decay of its infra-red absorption at 2048cm ~. We have been exploiting this to observe the onset of CO inhibition with time, and its dependence on concentration. SF-FTIR has also been used to monitor the time-dependence of the bands arising from CO bound to the FeMoco active site. It is known that use of deoxyATP (dATP) instead of ATP slows the reduction of protons and acetylene by nitrogenase by a factor of seven. By contrast, we find the low-CO band at 1904 cm -I peaks at 18s using dATP compared to 6s using ATP, and the high-CO band at 1936cm -~ peaks at the same time with dATP or ATP. Both bands are reduced about three-fold in intensity however. It is an aim of this project to stabilise the CO-nitrogenase complex for eventual crystallisation. Aluminium fluoride, together with ADP, is an ATP analogue that locks the MoFe and Fe protein together in a transition state complex. It is hoped that CO can also be locked into this complex. 1.
George, N.J., Ashby, G.A., Wharton, C.W., and Thorneley, R.N.F.J. Am. Chem. Soc. 119, 6450-6451 (1997)
The Biotechnology and Biological Sciences Research Council (BBSRC) is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
455
Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase Julia S. Thrower, Richard Blalock III, Judith P. Klinman Departments of Chemistry and of Molecular and Cell Biology; University of California, Berkeley; Berkeley, California 94720-1460, USA (e-mail.'[email protected]) 1-Aminocyclopropane-l-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2, and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by 02 and ascorbate. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado. We are currently examining the kinetics and chemical mechanism of a slower alternative substrate for ACC oxidase.
ket = v , , k exp[ - ( L + AG o)2 / 4 L R T ]
Electronic structure of the square-planar triplet ground state bis(biuretato) cobalt(III) compounds Peter W. Thulstrup, Erik Larsen Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, DENMARK (e-mail. pwt@kvl, dk) The extraordinary square-planar bis(biuretato) cobalt(III) coordination compounds, having a spin-triplet ground state, were discovered about 30 years ago I. This type of intermediate spin ground state compound provides an excellent model system for understanding the electronic structure of intermediate spin d 6 systems. Both phthalocyanine iron(II) and tetraphenylporphyrin iron(II), which are of obvious bioinorganic relevance, are known to have a spin-triplet ground state 2, but a full understanding of their electronic structure and it's biological relevance has never been obtained. In contrast to these iron(II) species, the bis(biuretato) cobalt(III) compounds provide a system with a simple ligand n-system as the framework for the cobalt(III) metal center. This combination makes experimental investigations both easier to perform and understand. The electronic structure of the anionic coordination compound shown to the right has been Pn Ph studied with absorption and circular dichroism spectroscopy in the NIR, UV-VIS and UV, including measurements in the vacuum UV region. Measurements of the magnetic susceptibility and the magnetic circular dichroism have also been performed. Furthermore, / o~_/_ \ ~ .~ a theoretical investigation, using ligand field and density functional theoretical calculations \ /\ / was carried out. The obtained results have been rationalized in terms of the electronic 0 structure of the bis(biuretato) cobalt(III) compounds, and they point to a 3d configuration of the type: (Z2) 2 (x2-y2) 2 (ZX) I (yz) I . 1. Birker, P.J.M.W.L., Bour, J.J., and Steggerda, J.J., Inorg. Chem., 12, 1254-1259 (1973) 2. Tanaka, K., Elkaim, E., Li, L., Jue, Z.N., Coppens, P., and Landrum, J., J. Chem. Phys., 84, 6969-6978 (1986)
456
Journal of Inorganic Biochemistry 86 (2001)
ENDOR studies of the ligation and structure of the non-heme iron site in ACC oxidase David L. Tiemey a, A m y M. Rocklin b, John D. Lipscombb, L awrence Que Jr. b and Brian M. Hoffman c C~Department of Chemistry, University of New Mexico, Albuquerque, NM87131, USA (e-mail: dtierney@unm, edu) bDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA CDepartment of Chemistry, Northwestern University, Evanston, IL 60208, USA The final step in ethylene biosynthesis, oxidation of 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene, 1s catalyzed by the Fe(II) dependent enzyme, ACC oxidase (ACCO). We present here a detailed 35 GHz pulsed and CW ENDOR examination of the coordination of Fe by ACCO in the absence and in the presence of substrate/inhibitor. Analysis of orientation-selective 14'15N and ~70 ENDOR data is interpreted in terms of a structural model of the ACCO active site for both states.
Calcium in bacterial peroxidases -pseudomonas stutzeri cytochrome c peroxidase Cristina G. Tim6teo a, Pedro Tavares a, Graham W. Pettigrew b and Isabel Moura a c~Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de Cidncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Capariea, Portugal h Department of Preclinical Veterinary Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall Edinburgh EH9 1QH, United Kingdom The Paracoccus '(Pa.) denitrificans cytochrome c peroxidase (CCP), one of the best studied bacterial CCP's is known to have a dependence for calcium of the activity, and two distinct calcium biding sites: site I, situated between the two domains of the monomer, and site II, situated in the dimer interface ~. Site II is thought to play a part in enzyme activation by promoting the low-spin to high-spin switch of peroxidatic heine and to promote the dimerization of the enzym e2. Pseudomonas (Ps.) stutzeri CCP was purified through three chromatographic steps and characterized biochemically and spectroscopically in the native, mixed valence and calcium incubated mixed valence forms. Two different ferrocytochromes were used as electron donors for Ps. stutzeri CCP activity studies: Ps. stutzeri cytochrome c55z, its physiological electron donor, and horse heart cytochrome e. The interaction of these cytochromes with Ps. stutzeri CCP is very different and they exhibit opposite activity dependence of salt concentration. The enzyme was isolated in a form where calcium site I is empty and calcium site II filled, w h i c h w a s not possible in Pa. denitrificans CCP because of the different calcium site afffmities. This form of Ps. stutzeri CCP is a dimer (unlike the CCP from Pa. denitrificans that is purified in dimer / monomer equilibrium), which confirms the role of calcium site II in the dimerization of CCP. The presence of calcium in site I doesn't seem to play a part in the activation, dimerization or activity of the enzyme. 1. Gilmour R., Goodhew C.F., Pettigrew G.W., Prazeres S., Moura J.J.G., Moura I., Biochem. J., 300, 907-914 (1994) 2. Gilmour R., Prazeres S., McGinnity D.F., Goodhew C.F., Moura J.J.G., Moura I., Pettigrew G.W., Eur. J. Biochem., 234, 878-886 (1995) The Fundaq~o para a Ci6ncia e Tecnologia (FCT-MCT) of Portugal is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
457
Analysis of the structural and electronic properties of new azide and fluoride mononuclear Mn(III) complexes by high frequency EPR C a r o l e D u b o c - T o i a a, C l a i r e M a n t e l a'b, M a r i e - N o , l i e C o l l o m b b, A n n e - L a u r e B a r r a a, A l i a H a s s a n a, A l a i n D e r o n z i e r b, J a c q u e s P 6 c a u t c
" Grenoble High M a g n e t i c F i e l d Laboratory, CNRS-MP1, BP 166, 38042 Grenoble Cedex 9, France(e-mail : [email protected]) b L a b o r a t o i r e d ' E l e c t r o c h i m i e O r g a n i q u e et de P h o t o c h i m i e Redox, 38041 Grenoble F r a n c e c L a b o r a t o i r e de Chimie I n o r g a n i q u e et Biologique, 38054 Grenoble Cedex, F r a n c e Our goal was to synthesize a series of mononuclear Mn(III) complexes with azide and fluoride ligands, anions that are known to inhibit metalloproteins containing manganese, in order to perform a study of their structural (X-ray) and electronic (high frequency EPR: HF-EPR) properties. Among these enzymes the active site of the manganese superoxide dismutase is a mononuclear Mn(III) ~ , . . e , ~ complex. The study of the interaction of potential ligands with the manganese site is often difficult due to the lack of effective spectroscopic methods like classic EPR (X-band, 9 GHz) (Mr.(III), S = 2). The technique of high frequency EPR (HF-EPR) has proven to be effective in elucidating the electronic structure of integer spin compounds, also called non-Kramers systems. We will present a complete study of a series of complexes that were structurally characterized by X-ray ~. Here is represented the structure of [Mn(terpy)F3] (terpy : 2,2':6'2"-terpyridine). A comparative analysis will be done to define the correlation that we observed between the structural properties of the complexes and the electronic parameters issued from the HF-EPR study. 1. Limburg J., Vrettos J. S., Crabtree R, H., Brudvig G. W., de Paula J. C., Hassan A., Barra A.L., Duboc-Toia C., Collomb M.-N. Inorg. Chem., 40, 1698-1703 (2001).
Stopped-flow time-resolved infra-red spectroscopy of the activation of small molecules by nitrogenase John D. Tolland, Gillian A. Ashby, Simon J. George, Roger N.F. Thorneley Biological C h e m i s t r y Dept. John lnnes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH (e-mail: j o h n . r o l l a n d @ b b s r c . a c , uk) We have been using stopped-flow Fourier transform infra-red spectroscopy (SF-FTIR) to monitor the dynamics of inhibition of nitrogenase by carbon monoxide. SF-FTIR gives the first real-time measurement of consumption of a substrate, azide, through decay of its infra-red absorption at 2048crn -~. We have been exploiting this to observe the onset of CO inhibition with time, and its dependence on concentration. SF-FTIR has also been used to monitor the time-dependence of the bands arising from CO bound to the FeMoco active site. It is known that use of deoxyATP (dATP) instead of ATP slows the reduction of protons and acetylene by nitrogenase by a factor of seven. By contrast, we find the low-CO band at 1904 cm ~ peaks at 18s using dATP compared to 6s using ATP, and the high-CO band at 1936cm l peaks at the same time with dATP or ATP. Both bands are reduced about three-fold in intensity however. It is an aim of this project to stabilise the CO-nitrogenase complex for eventual crystallisation. Aluminium fluoride, together with ADP, is an ATP analogue that locks the MoFe and Fe protein together in a transition state complex. It is hoped that CO can also be locked into this complex. 1.
George, S.J., Ashby, G.A., Wharton, C.W., and Thomeley, R.N.F.J. Am. Chem. Soc. 119, 6450-6451 (1997)
The Biotechnology and Biological Sciences Research Council (BBSRC) is acknowledged for financial support.
458
Journal of Inorganic Biochemistry 86 (2001)
Preparation of o x o v a n a d i u m ( I V ) Schiff base complexes derived from aromatic o-hidroxyaldehydes and simple sugars containing amino groups k T o m a z , J. C o s t a P e s s o a , I. C o r r e i a and S. M a r c ~ o
Centro ae Quimica EstruturaI, Instituto Superior T~cnico, Av. Rovisco Pais, 1049-001 Lisbon PORTUGAL (e-mail: [email protected], utl.pt) There has been great effort in the past years to find efficient insulin-mimetic vanadium complexes. We are extending our studies towards organism-friendly non-toxic ligands such as sugars and sugar derivatives. The present communication deals with the preparation of several complexes formulated as [VO(sal-D-gcsN)(H20)2], [VO(sal-D-galacN)(H20)] and [V203(sal-D-glucN)(H20)], in which the ligands are Schiff bases derived from the condensation of salicylaldehyde (sal) and D(+)-Glucosamme (D-gcsN), D(+)-Galactosamine (D-galacN) and DGlucamine (D-glucN). A light-green complex formulated as [VO(pyr-D-glucN)(HeO)].X* was also prepared where pyr-D-glucN is the Schiff base derived from pyridoxal and D-glucamine, and X ÷ is Na ÷ or K*. The complexes were characterized by elemental analysis, IR, EPR, UV-VIS and Circular Dichroism spectroscopy. The preparation of complexes with the corresponding reduced Schiffbase complexes is also explored. We thank Fundo Europeu para o Desenvolvimento Regional and Funda~o para a Ci~ncia POCTI/35368/Qui/2000 and BD/18548/98 fellowship) for financial support.
e Tecnologia (project
Structural and spectroscopic characterization of new Cu(II)-dipeptide complexes Maria H. Torrea, Gianella Facchin a, Eduardo Kremera, Enrique J. Baranb, Oscar E. Piroc aQuimica Inorgdzniea, D.E.C., Facultad de Quimica, UDELAR, Gral. Flores 212, 11800, Montevideo, Uruguay, (e-mail [email protected]) bCEQUINOR; CDepartamento de Fisica and IFLP, Facultad de Ciencias Exactas, UNLP, I900, La P/ata, Argentina Cu(II) complexes containing peptides as ligands are often good model systems to obtain a better insight into the characteristics of naturally occurring copper proteins. As a part of our study of a series of copper compounds with peptides two new Cu(II) complexes of stoichiometry [Cu(L-dipeptide)] containing L-AIa-L-Ile and LAla-L-Thr were prepared and characterized by single crystal X-ray diffractometry, IR i~ e.4 S and electronic spectroscopy. Results were compared with those of Cu-L-Ala-L-Val and Cu-L-AIa-L-Phe previously studied, i In the four complexes the Cu(II) is at the square base center of an elongated pyramidal environment, equatorially coordinated by a dipeptide molecule acting as a tridentate ligand as shown in the figure. The equatorial copper coordination gives rise to polymeric structures in the lattice. Superoxide dismutase (SOD)-like activity was also tested. The values of ICs0, using the nitrobluetetrazoliurn/superoxide reduction assay were 3.3 E-4 M (CuL-Ala-L-Ile) and 9.9 E-5 M (CuL-Ala-L-Thr). 1.
G. Facchin, M. H. Torre, E. Kremer, E. J. Baran, O. Piro, Z. Naturforsch, 55b, 1157 - 1162 (2000)
Journal of Inorganic Biochemistry 86 (2001)
Studies
o n E u II
459
chelates in relation to magnetic esonance imaging
t~va T 6 t h , L f i s z l 6 B u r a i , A n d r 6 E. M e r b a c h
Institute of Inorganic and Analytical Chemistry, University of Lausanne, BCH, 1015 Lausanne, Switzerland (e-mail. Eva.Jakab- Toth@icma. unil. ch) TETA ODDM DOTA ODDA
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The recent interest in ~:he aqueous solution chemistry of Eu II chelates has been prompted by two main aspects. L First, Eu ~ is isoelectronic with Gd m, therefore studying relaxation phenomena on the Eu H analogues can give further insight into the mechanisms that govern relaxation on Gd III complexes as well and thus help the development of novel MRI contrast agents. On the other hand, one can take advantage of the redox abilities of Eu ~z and design EuH/Eu ~ based responsive MRI contrast agents which are sensitive to the redox state of the biological environment. We will present recent results related to these aspects. 2.
Toth E., Burai L. and Merbach A.E., Coord. Chem. Reviews, (2001) in press.
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E vs AgZAgCI{V)
Fig. 1. The redox potential of Eull/Eu 111 changes substantially as a function of the ligand
Density functional description of the spin states involved in the early stages of the dioxygenation of [(MeC(CH2PPhz)3)Ir(cathecolate)]+: a possible model of dioxygenases Federico Tottia, Alessandro Bencinia, Andrea Scozzafavab "Dipartimento di Chimica, Universith di Firenze, Via Maragliano 75/77, 50144, Firenze ( I t a l y ) (e-mail. [email protected], i 0 ~Dipartimento di Chimica, Laboratorio di Bioinorganica, Universitgt di Firenze, Via Capponi 7, 50121, Firenze (Italy) The earlier part of' our work we have been applied to, has been devoted to the study o f the basic points related to the reaction path involving the [(triphos) M(cathecolate)] + + 02 (M = Co(III) and Ir(III)) moieties in their fundamental states. Nevertheless, the above reactions are spin-forbidden, and the spin-orbit coupling is supposed to be the most probable mechanism that can allow the reactions. In order to have an insight in such a topic, we have carried out calculations on the excited states for the already found stationary points, plus some others on different potential surfaces. In this framework we have used both pure density functional approach (VWN + Stoll correction) with slater-type basis sets (ADF, Amsterdam Density Functional package) and a hybrid one (B3LYP) with gaussian-type basis sets (Gaussian98 package). As regards the [(triphos)Ir(III)(cathecolate)]+ + 02 reaction, we have attempted to scan the potential surfaces for the low lying spin states possibly involved in the reaction, that is 3A state and the two possible ~A states. The importance of the mono-protonated cathecol binding in the extra-diol cleavage has been also investigated following the same computational protocol used for the intra-diol dioxygenases. In this case we have taken into consideration the [(triphos)Ir(III)(cathecolate)H]2+ + O2 reaction. Single points solvent calculations (IPCM) have been also performed for several geometries with Gaussian98. A new plausible mechanism for the intra- and extra-dioxygenases has been then suggested supported by the computed data obtained in this work.
460
Journal of Inorganic Biochemistry 86 (2001)
Dinuclear manganese complexes as structural and spectroscopic models for manganese catalases Michael U. Triller a, Vincent L. Pecoraro b, Annette Rompel a and Bernt Krebs a " Anorganisch-Chemisches Institut der Westffilischen Wilhelms- Universitgit, Wilhelm-Klemm-Str. 8. 48149 Miinster, Germany, b Department of Chemistry, University of Michigan, 930 North University Boulevard, Ann Arbor, Michigan 48109-1055, U,S.A. Manganese catalase from T. thermophilus (TTC) catalyzes the disproportionation of H202 to H20 and O2.1 Four oxidation states of the enzyme have been characterized, namely the catalytically active (2,2) and 2_ (3,3) states and the inactive (2,3) and (3,4) states. I The dinuclear complexes [Mn(L)(,u-OAc)]2[CIO4]2 1, [Mn2(L)2(/.t-O)(/.t-OAc)][ClO4]3 2 and [Mn(L)(,u-O)]2[C104]2[PF6] 3 (L = Bis(picolyl)((l-methylirnidazol-2-yl)methyl)amine) are structural and spectroscopic models for the (2,2), (3,3) and (3,4) states, respectively. Like the (2,2) form of TTC, a UV/Vis spectrum of 1 shows no absorption in the visible region, while 2_ exhibits a band at 495 nm (492 nm for the (3,3) form of TTC). I EPR measurements reveal a 16 line spectrum for 3. Complexes 1, 2 and 3 disproportionate H202. Attempts to synthesize model complexes for the chloride inhibited form of TTC yielded [Mn(L)(C1)2] [CIO]44 and [(Mn(L)(C1))2~-O)][C104]2 5_.-' 3. 4.
Whittaker M.M., Barynin V.V., Antonyuk S.V., Whittaker J.W., Biochemistry, 38, 9126 - 9136 (1999) Antonyuk S.V., Melik-Adamyan V.R., Popov A.N., Lamzin V.S., Hempstead P.D., Harrison P.M., Artymyuk P.J., Barynin V.V., Crystallography Reports, 45, 111-122 (2000)
pH dependent Hg(II) coordination in metallothioneins: a 199mHg TDPAC study W. Tr6gera, B. Ctorteckaa, P. Fallerb, M. Va~b.kb and
the I S O L D E Collaboration c
alnstitute of Experimental Physics II, University of Leipzig, Linn~strafle 5, 04103 Leipzig, Germany (email. troeger@physik, uni-leipzig.de) blnstitute of Biochemistry, University of Ziirich, Winterthurerstr. 190, 8057 Ziirich, Switzerland ~CERN 1211 Geneva. Switzerland 7 Mammalian metallothioneins (MT) are ubiquitous, 08 o,oo~ ~t cysteine-rich proteins of low molecular weight which bind dl 0 o,6 Hg'-MT •~.4 ii pH 8.6 metals ions such as Zn(II), Cd(II), Cu(I) and Hg(II) in metalthiolate clusters. They play an important role in the metabolism and in the modulation of the essential trace element zinc and copper and in the binding of toxic heavy ~6 ~ Hg'-MT metals. The latter suggests also the involvement in cellular ~os[" '1"7 -6.4! , pH 2 detoxification mechanisms. Several 3D structures have been 02~, I 'w' ~. • °'° - J v"~,'-,~,q',~h..,~.~ solved for mammalian Me(II)7-MT, containing Zn(II) and/or -o.o5 L. i Cd(II) ions. These metal ions are tetrahedrally coordinated by o 2 4 6 B 10 12 o 5 lO 15 20 both bridging and terminal thiolates in cluster structures. Here, Frequency [Grad/s] Time [ns] we report on Hg(II) coordination in rabbit liver MT by time Fig 1: TDPAC time spectra (left) and thei Fourier transforms differential perturbed angular correlation (TDPAC) (right) of Hg7-MT at pH 8.6 (top) and pH 2 (bottom). The low frequencyat pH 8.6 indicates a tetrahedral and the high frequency spectroscopy. With TDPAC the local environment of the at pH2 a diagonal Hg coordinationgeometry TDPAC probe, here 199rnHg, can be studied by the correlation of two subsequent gamma quanta emitted by the probe• The titration of apo-MT with Hg(II) (1-7 equiv.) revealed mainly two- and fourfold Hg(II) coordinations depending on Hg(II) concentration and pH. At pH 2.0 twofold coordinations dominate whereas at pH 8.6 also higher coordination numbers occur.
)'01
451
Copper complexes of 1,2,4-triazolines derivatives of c~-amino acids M. Tegoni a, M. Cingi Biagini a, E. Ferloni ~, M. Lanfranchi a, M.A. Pellinghelli a, "Dipartimento di Chimica Generale ed Inorganica, University o f Parma, Parco Area delle Scienze, 17A, 43100 Parma, I T A L Y (e-mail: [email protected], unipr, it) The study of model systems of metalloenzymes containing copper(II) is of great importance in order to understand the mechanisms of action of these proteins. Here we present the synthesis and the characterization both in solid and in solution state of new triazoline derivatives functionalized with amino acid residues and their copper(II) complexes. Ligands 1 and 2 have been synthesized by double condensation of thiocarbonohydrazide with BOC-protected L-amino acids activated on the carboxylic group followed by H N--N deprotection of the amino group. Both ligands 1 and 2 have been characterized in solution / \\ .NHa by potentiometric studies. In the case of ligand 1 the determined pKa were 6.48(1) and S 8.03(1). Copper (II) complexes with ligand 1 have been isolated and crystallized both in the N I 2 R ratio of M:L 1:1 and 1:2 and their structure determined. In all the complexes the ligand NH showed to be protonated on the amino group and coordinated to the metal ion via hydrazinic nitrogen and sulfur atoms. The complex [CuC12(1)]CI presents a square planar coordinated Ligand 1: R = H copper ion, whereas in [Cu(OH2)2(1)z](SO4)2 the copper atom is octahedrally coordinated Ligand 2: R = CH3 with two water molecules in axial position. EPR and MAGSUS experiments on the complex [CuCI2(1)]C1 in the solid state revealed an anti ferromagnetic behaviour with an inversion temperature of 19(1) K. This behaviour is most probably due to weak interactions between copper atoms and sulfur atoms of adjacent molecules, forming polymeric Cu - S - Cu chains. +
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Synthesis and characterization in solution of hydroxamic acid derivatives as new potential enzyme inhibitors M. Tegoni ", F. Dallavalle", M.A. Santos b "Dipartimento di Chimica Generale ed lnorganica, Chimica Analitica, Chimica Fisica, University of Parma, Parco Area delle Scienze, 17A, 43100 Parma, Italy. ~'Centro de Quimica Estrutural, Complexo I, Instituto Superior Tkcnico, 1049-001 Lisboa-codex, Portugal. (e-mail: masantos@popsrv, ist. utl.pO The development of suitable amino acids or short peptides containing hydroxamate groups are of current interest because they can act as inhibitors for a large number of metalloenzymes. Herein we present two new potential metalloenzyme inhibitors L1 and L2 (H2L). They 0 Ph incorporate one hydroxamate and one monopeptide-carboxylate group at terminal L1 HO. fl]~ / ~ .~L ~J positions for metal binding and enzyme-inhibitor interaction, respectively. Besides ~- v -,f - , , f o COOH the synthesis of the compounds, we report the equilibrium studies with Cu 2+, Ni 2+ and Zn 2+ The preparation of the ligands required standard protecting-group o techniques and it mainly involved the connection of the two molecular units, L2 H O ' - ~ / I vI ~ / ~ ]~- S - .,() through a peptide bond, followed by formation of the hydroxamic acid. Complex formation studies with this set of metal ions revealed the existence of the o COOH corresponding ML, ML2 and ML2H_t species. In the case of Ni z+, the ML3 species was also found. All the calculated constants are very close to those determined for monohydroxamic acids. In the case of copper(lI), the solution containing CuL2 and CuLzH.I are green 0q~,~ ca. 630 run), showing that the coordination mode is a 2 x {0,0} chelation on the hyrdoxamate group. The species CuL2 has most probably a planar coordination geometry, whereas in ZnL2 the metal is probably tetrahedral. ML2H.j species are most probably deprotonated on one hydroxamate nitrogen atom, rather than forming an hydroxo specie with an OH- coordinated in an axial position. The authors thank to the COST D21 program for financial support.
452
Journal of lnorganic Biochemistry 86 (2001)
The interaction of the substrate analoguep-nitrophenol with the oxidised type-3 copper protein tyrosinase studied by paramagnetic NMR A . W . J . W . T e p p e r a, L . B u b a c c o b, M . U b b i n k a, U . K o l c z a k a G . W . C a n t e r s a
'%eiden institute of Chemistry, Leiden University, Einsteinweg 55, 2300RA Leiden, The Netherlands bDepartment of Biology, University of Padua, Via Trieste 75, 30121 Padua, Italy We have recently shown that the oxidised [Cu(II)-R-Cu(II)] form of the type-3 copper protein tyrosinase (Tymet) from Streptomyces antibioticus is amenable to study by paramagnetic NMR methods, providing a sensitive probe of the active site coordination [1]. The binding of exogenously added inhibitors leads to drastic changes in the paramagnetic part of the 1H NMR spectra. Here we report on a paramagnetic NMR study of the interaction of the monodendate suhstrate analogue p-nitrophenol with native Tymet and its halide bound derivatives. A titration of Tymet with p-nitrophenol showed that the electronic active site smacture remains largely unperturbed upon binding of the ligand. Titrations of the F- and C1- bridged Cu2 derivatives of Tymet with p-nitrophenol induced substantial changes in the paramagnetic ~H spectra in terms of Hisproton signal positions and linewidths. The ligand appeared to be in fast exchange with either TymetF or TymetC1. In both cases, three sharp HisN a proton signals appeared more sensitive to the presence ofp-nitrophenol than the other remaining three, possibly indicating that the monophenol interacts more strongly with one copper ion in the dinuclear site. The nitrophenol / Tymet exchange process appeared to induce significant broadening of the 2 diamagnetic p-nitrophenol ring proton signals in the bulk. Determination of both T~ and T2 relaxation enhancements of the bulk nitrophenol signals in samples containing various [nitrophenol]/[Tym~t] allowed estimation of the ligand dissociation constant Kd and the longitudinal and transversal paramagnetic relaxation rates of Tymet bound nitrophenol, showing that the monophenol is directly coordinated to copper through the hydroxyl moiety. The relevancy of the findings for understanding the catalytic mechanism is discussed. 1. Bubacco, L., Salgado, J., Tepper, A.W.J.W., Vijgenboom, E., Canters, G.W., FEBS Letters 442, 215-220, 1999.
A novel class of pH-sensitive paramagnetic CEST contrast agents for MRI applications Enzo Terrenoa,SilvioAime a, Alessandro Bargea, Daniela Delli Castellia, Flemming Ulrich Nielsenb ~' Dipartimento di Chimica 1FM, Universit~ di Torino, Via P. Giuria 7, I-10125, Torino, Italy Laboratorio lntegrato di Metodologie Avanzate, Bioindustry Park Canavese, Via Ribes 5, 110010, Colleretto Giacosa (TO),Italy, (e-mail: [email protected]) The recently introduced new class of Contrast Agents based on Chemical Exchange Saturation Transfer (CEST) may have a huge potential for the development of novel applications in the field of MRI. Many diamagnetic small organic molecules (aminoacids, sugars, heterocyclic compounds) as well as macromolecular systems (dendrimers and polymers) have been so far investigated. 1 A good CA for CEST application has to possess mobile protons whose exchange rate with water is as high as possible before coalescence takes place. This means that larger shift separation between the two exchanging ~ ~.N ~.;~'/"<° ~ sites results in an enhanced CEST effect. Recently, Sherry et al. showed that a particularly o:j[,..~ useful source of highly shifted exchangeable protons can be provided by the slowly exchanging .~ %j~o~ water protons bound to a paramagnetic Eu(III)-chelate. 2 An alternative source may be represented by labile protons on the ligand of paramagnetic complexes. In this contribution, we present the properties of a series of Ln(III)-chelates of a tetraglycineamide derivative of DOTA, which contains four equivalent labile amidic protons in close proximity of the paramagnetic center (see figure). The best CEST effect has been obtained for the Yb(III) derivative. Interestingly, the effect is markedly pH-dependent. A ratiometric method for the "in vivo" pH measurement has been developed which allows to observe a CEST effect not longer dependent on the absolute concentration of the CA. 1. Ward K.M., Aletras A.H. and Balaban R.S., J. Magn. Res., 143, 79-87, 2000. 2. Zhang S., Winter P., Wu K., Sherry A.D., J. Am. Chem. Soc., 123, 1517-1518, 2001.
Journal of lnorganic Biochemistry 86 (2001)
453
NMR detection of the inner-rotatiopn of water around its coordination axis in Eu(III) complexes Enzo Terreno a, Silvio Aime, Alessandro Barge, Mauro Botta, Erik Bruno Dipartimento di Chimica IFM, Universitd di Torino, Via P. Giuria 7,1-10125, Torino, Italy The huge amount of work carried out in the last decade in the field of Lanthanide(llI) complexes has provided an in-depth understanding of their solution structures and dynamics. In particular it has been possible to get a good understanding of the main determinants of the exchange rates (kex) of the coordinated water molecule. On the basis of ~70-NMR investigations on a large number of coordination schemes, it has been found that the exchange lifetime rM (1/kex) c o v e r s almost 6 order of magnitude (fi'om l0 -9 to 1 0 -3 S!). 120 ~o.~ The cationic tetrabenzylglycinamide-DOTA Eu(lll) complex (Eu-DOTAM-GIyBz) owns ,0~ . ~ , ~ ~ , one of the longest zTv.,(0.59 ms at 298K). Thus, on this system, it has been possible to ~0measure the relaxation time T ~ of the resonance of the coordinated water over an~ ~01 extended range of temperature (solvent CD3CN) and to compare the observed behaviour ~ ~ ! with that of 1H-resonances of the macrocyclic ligand. The analysis of the obtained I/Tt ~,,i "'!'" ° : " i vs temperature profiles allows to assess that the inner rotation of the coordinated water ~ I 0[ . . . . . --occurs on a faster timescale than the overall reorientation of the complex. Analogous ~ _0 _,~, ~0 ~0 3o0 ~0 ~ 360 results were obtained on related Eu(llI) complexes. J- K On the basis of the close analogy between Eu(III) and Gd(lll) complexes, this finding suggests that a new item has to be considered in the design of contrast agents endowed with enhanced relaxivities. .
Electron tunneling in cytochrome c crystals F. Akif Tezcark Brian R. Crane, Jay R. Winkler, Harry B. Gray Beckman Institute, California Institute of Technology, Pasadena, CA 91125, U.S.A. The factors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the very short time that tunneling occurs. In order to circumvent this structural ambiguity, we have measured and analyzed the kinetics of electron 1013 Electron Tunneling transfer (ET) b e t w e ~ native and Zn-substituted tuna cytochrome c (cyt c) ps Timetable molecules in crystals of known structure (1.5 ~. resolution). ET rates (320 s 1011 10 lo for *Zn-cyt c --+ Fe(lll)-cyt c; 2000 s-' for Fe(II)-cyt c --~ Zn-cyt c +) over a ZnFe distance of 24.1 A closely match those for intraprotein electron tunneling nS ~> protein over similar donor-acceptor separations. These results indicate that van der 10 -7 ~[3 [3 . time Ms ,/~a v ~ o o Waals interactions and water-mediated hydrogen bonds are effective coupling elements for tunneling across a protein-protein interface. Zn-porphyrin lO ~ eg photoreactions should be particularly useful for the study of ET in other heme ms lO ~ protein crystals, as well as for the generation of short-lived redox intermediates io-i that are amenable to structural characterization. S vac~Jum H~O | ,
This project was supported by the Arnold and Mabel Beckman Foundation, the National Science Foundation and the Helen Hay Whitney Foundation.
10
20
distance, ~k
,
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30
Journal of inorganic Biochemistry 86 (2001)
454
Kinetic and equilibria studies of the aquation of the trinuclear platinum anticancer agent BBR3464 Donald S. Thomasa, Murray S. Davies a, Alexander Hegrnansb, Susan J. Berners-Pricea, Nicholas Farrellb "Department of Chemistry, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009 AUSTRALIA. (e-mail. [email protected], edu.au) ~Department of Chemistry, Virginia Commonwealth University, 1001 Main Street, Richmond, Virginia, 23284-2006, USA. BBR3464, which breaks the old barriers of activity predicated by cisplatin, is providing a fresh perspective in the field of platinum anti-cancer drugs. [1H,15N] HSQC NMR spectroscopy has been used to investigate the hydrolysis profile of BBR3464 under conditions of different ionic strength and temperature and in the presence of phosphate. Rate and equilibrium constants for the aquation of BBR3464 have been determined, together with the acid dissociation constants for the aquated species. The equilibrium lies further towards the aquated species than occurs for the related dinuclear complex 1,1/t,t (n=6) ~ under the same conditions, while the pKa value(s) of the aquated drug is identical (5.62). The presence of phosphate does not slow the initial aquation of BBR3464 but reversible reaction between aquated species and phosphate does occur.
1. Davies, M. S., Cox, J. W., Bemers-Price, S. J., Barklage, W., Qu, Y and Farrell, N., Inorg. Chem., 39, 1710-1715 (2000). The authors thank the Australian Research Council, U.S. National Institute of Health, U.S. National Science Foundation and the American Cancer Society for financial support.
Stopped-flow time-resolved infra-red spectroscopy of the activation of small molecules by nitrogenase John D. Tolland, Gillian A. Ashby, Simon J. George, Roger N.F. Thorneley Biological Chemistry Dept. John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH (e-mail: [email protected]) We have been using stopped-flow Fourier transform infra-red spectroscopy (SF-FTIR) to monitor the dynamics of inhibition of nitrogenase by carbon monoxide. SF-FTIR gives the first real-time measurement of consumption of a substrate, azide, through decay of its infra-red absorption at 2048cm ~. We have been exploiting this to observe the onset of CO inhibition with time, and its dependence on concentration. SF-FTIR has also been used to monitor the time-dependence of the bands arising from CO bound to the FeMoco active site. It is known that use of deoxyATP (dATP) instead of ATP slows the reduction of protons and acetylene by nitrogenase by a factor of seven. By contrast, we find the low-CO band at 1904 cm -I peaks at 18s using dATP compared to 6s using ATP, and the high-CO band at 1936cm -~ peaks at the same time with dATP or ATP. Both bands are reduced about three-fold in intensity however. It is an aim of this project to stabilise the CO-nitrogenase complex for eventual crystallisation. Aluminium fluoride, together with ADP, is an ATP analogue that locks the MoFe and Fe protein together in a transition state complex. It is hoped that CO can also be locked into this complex. 1.
George, N.J., Ashby, G.A., Wharton, C.W., and Thorneley, R.N.F.J. Am. Chem. Soc. 119, 6450-6451 (1997)
The Biotechnology and Biological Sciences Research Council (BBSRC) is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
455
Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase Julia S. Thrower, Richard Blalock III, Judith P. Klinman Departments of Chemistry and of Molecular and Cell Biology; University of California, Berkeley; Berkeley, California 94720-1460, USA (e-mail.'[email protected]) 1-Aminocyclopropane-l-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2, and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by 02 and ascorbate. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado. We are currently examining the kinetics and chemical mechanism of a slower alternative substrate for ACC oxidase.
ket = v , , k exp[ - ( L + AG o)2 / 4 L R T ]
Electronic structure of the square-planar triplet ground state bis(biuretato) cobalt(III) compounds Peter W. Thulstrup, Erik Larsen Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, DENMARK (e-mail. pwt@kvl, dk) The extraordinary square-planar bis(biuretato) cobalt(III) coordination compounds, having a spin-triplet ground state, were discovered about 30 years ago I. This type of intermediate spin ground state compound provides an excellent model system for understanding the electronic structure of intermediate spin d 6 systems. Both phthalocyanine iron(II) and tetraphenylporphyrin iron(II), which are of obvious bioinorganic relevance, are known to have a spin-triplet ground state 2, but a full understanding of their electronic structure and it's biological relevance has never been obtained. In contrast to these iron(II) species, the bis(biuretato) cobalt(III) compounds provide a system with a simple ligand n-system as the framework for the cobalt(III) metal center. This combination makes experimental investigations both easier to perform and understand. The electronic structure of the anionic coordination compound shown to the right has been Pn Ph studied with absorption and circular dichroism spectroscopy in the NIR, UV-VIS and UV, including measurements in the vacuum UV region. Measurements of the magnetic susceptibility and the magnetic circular dichroism have also been performed. Furthermore, / o~_/_ \ ~ .~ a theoretical investigation, using ligand field and density functional theoretical calculations \ /\ / was carried out. The obtained results have been rationalized in terms of the electronic 0 structure of the bis(biuretato) cobalt(III) compounds, and they point to a 3d configuration of the type: (Z2) 2 (x2-y2) 2 (ZX) I (yz) I . 1. Birker, P.J.M.W.L., Bour, J.J., and Steggerda, J.J., Inorg. Chem., 12, 1254-1259 (1973) 2. Tanaka, K., Elkaim, E., Li, L., Jue, Z.N., Coppens, P., and Landrum, J., J. Chem. Phys., 84, 6969-6978 (1986)
456
Journal of Inorganic Biochemistry 86 (2001)
ENDOR studies of the ligation and structure of the non-heme iron site in ACC oxidase David L. Tiemey a, A m y M. Rocklin b, John D. Lipscombb, L awrence Que Jr. b and Brian M. Hoffman c C~Department of Chemistry, University of New Mexico, Albuquerque, NM87131, USA (e-mail: dtierney@unm, edu) bDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA CDepartment of Chemistry, Northwestern University, Evanston, IL 60208, USA The final step in ethylene biosynthesis, oxidation of 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene, 1s catalyzed by the Fe(II) dependent enzyme, ACC oxidase (ACCO). We present here a detailed 35 GHz pulsed and CW ENDOR examination of the coordination of Fe by ACCO in the absence and in the presence of substrate/inhibitor. Analysis of orientation-selective 14'15N and ~70 ENDOR data is interpreted in terms of a structural model of the ACCO active site for both states.
Calcium in bacterial peroxidases -pseudomonas stutzeri cytochrome c peroxidase Cristina G. Tim6teo a, Pedro Tavares a, Graham W. Pettigrew b and Isabel Moura a c~Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de Cidncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Capariea, Portugal h Department of Preclinical Veterinary Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall Edinburgh EH9 1QH, United Kingdom The Paracoccus '(Pa.) denitrificans cytochrome c peroxidase (CCP), one of the best studied bacterial CCP's is known to have a dependence for calcium of the activity, and two distinct calcium biding sites: site I, situated between the two domains of the monomer, and site II, situated in the dimer interface ~. Site II is thought to play a part in enzyme activation by promoting the low-spin to high-spin switch of peroxidatic heine and to promote the dimerization of the enzym e2. Pseudomonas (Ps.) stutzeri CCP was purified through three chromatographic steps and characterized biochemically and spectroscopically in the native, mixed valence and calcium incubated mixed valence forms. Two different ferrocytochromes were used as electron donors for Ps. stutzeri CCP activity studies: Ps. stutzeri cytochrome c55z, its physiological electron donor, and horse heart cytochrome e. The interaction of these cytochromes with Ps. stutzeri CCP is very different and they exhibit opposite activity dependence of salt concentration. The enzyme was isolated in a form where calcium site I is empty and calcium site II filled, w h i c h w a s not possible in Pa. denitrificans CCP because of the different calcium site afffmities. This form of Ps. stutzeri CCP is a dimer (unlike the CCP from Pa. denitrificans that is purified in dimer / monomer equilibrium), which confirms the role of calcium site II in the dimerization of CCP. The presence of calcium in site I doesn't seem to play a part in the activation, dimerization or activity of the enzyme. 1. Gilmour R., Goodhew C.F., Pettigrew G.W., Prazeres S., Moura J.J.G., Moura I., Biochem. J., 300, 907-914 (1994) 2. Gilmour R., Prazeres S., McGinnity D.F., Goodhew C.F., Moura J.J.G., Moura I., Pettigrew G.W., Eur. J. Biochem., 234, 878-886 (1995) The Fundaq~o para a Ci6ncia e Tecnologia (FCT-MCT) of Portugal is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
457
Analysis of the structural and electronic properties of new azide and fluoride mononuclear Mn(III) complexes by high frequency EPR C a r o l e D u b o c - T o i a a, C l a i r e M a n t e l a'b, M a r i e - N o , l i e C o l l o m b b, A n n e - L a u r e B a r r a a, A l i a H a s s a n a, A l a i n D e r o n z i e r b, J a c q u e s P 6 c a u t c
" Grenoble High M a g n e t i c F i e l d Laboratory, CNRS-MP1, BP 166, 38042 Grenoble Cedex 9, France(e-mail : [email protected]) b L a b o r a t o i r e d ' E l e c t r o c h i m i e O r g a n i q u e et de P h o t o c h i m i e Redox, 38041 Grenoble F r a n c e c L a b o r a t o i r e de Chimie I n o r g a n i q u e et Biologique, 38054 Grenoble Cedex, F r a n c e Our goal was to synthesize a series of mononuclear Mn(III) complexes with azide and fluoride ligands, anions that are known to inhibit metalloproteins containing manganese, in order to perform a study of their structural (X-ray) and electronic (high frequency EPR: HF-EPR) properties. Among these enzymes the active site of the manganese superoxide dismutase is a mononuclear Mn(III) ~ , . . e , ~ complex. The study of the interaction of potential ligands with the manganese site is often difficult due to the lack of effective spectroscopic methods like classic EPR (X-band, 9 GHz) (Mr.(III), S = 2). The technique of high frequency EPR (HF-EPR) has proven to be effective in elucidating the electronic structure of integer spin compounds, also called non-Kramers systems. We will present a complete study of a series of complexes that were structurally characterized by X-ray ~. Here is represented the structure of [Mn(terpy)F3] (terpy : 2,2':6'2"-terpyridine). A comparative analysis will be done to define the correlation that we observed between the structural properties of the complexes and the electronic parameters issued from the HF-EPR study. 1. Limburg J., Vrettos J. S., Crabtree R, H., Brudvig G. W., de Paula J. C., Hassan A., Barra A.L., Duboc-Toia C., Collomb M.-N. Inorg. Chem., 40, 1698-1703 (2001).
Stopped-flow time-resolved infra-red spectroscopy of the activation of small molecules by nitrogenase John D. Tolland, Gillian A. Ashby, Simon J. George, Roger N.F. Thorneley Biological C h e m i s t r y Dept. John lnnes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH (e-mail: j o h n . r o l l a n d @ b b s r c . a c , uk) We have been using stopped-flow Fourier transform infra-red spectroscopy (SF-FTIR) to monitor the dynamics of inhibition of nitrogenase by carbon monoxide. SF-FTIR gives the first real-time measurement of consumption of a substrate, azide, through decay of its infra-red absorption at 2048crn -~. We have been exploiting this to observe the onset of CO inhibition with time, and its dependence on concentration. SF-FTIR has also been used to monitor the time-dependence of the bands arising from CO bound to the FeMoco active site. It is known that use of deoxyATP (dATP) instead of ATP slows the reduction of protons and acetylene by nitrogenase by a factor of seven. By contrast, we find the low-CO band at 1904 cm ~ peaks at 18s using dATP compared to 6s using ATP, and the high-CO band at 1936cm l peaks at the same time with dATP or ATP. Both bands are reduced about three-fold in intensity however. It is an aim of this project to stabilise the CO-nitrogenase complex for eventual crystallisation. Aluminium fluoride, together with ADP, is an ATP analogue that locks the MoFe and Fe protein together in a transition state complex. It is hoped that CO can also be locked into this complex. 1.
George, S.J., Ashby, G.A., Wharton, C.W., and Thomeley, R.N.F.J. Am. Chem. Soc. 119, 6450-6451 (1997)
The Biotechnology and Biological Sciences Research Council (BBSRC) is acknowledged for financial support.
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Journal of Inorganic Biochemistry 86 (2001)
Preparation of o x o v a n a d i u m ( I V ) Schiff base complexes derived from aromatic o-hidroxyaldehydes and simple sugars containing amino groups k T o m a z , J. C o s t a P e s s o a , I. C o r r e i a and S. M a r c ~ o
Centro ae Quimica EstruturaI, Instituto Superior T~cnico, Av. Rovisco Pais, 1049-001 Lisbon PORTUGAL (e-mail: [email protected], utl.pt) There has been great effort in the past years to find efficient insulin-mimetic vanadium complexes. We are extending our studies towards organism-friendly non-toxic ligands such as sugars and sugar derivatives. The present communication deals with the preparation of several complexes formulated as [VO(sal-D-gcsN)(H20)2], [VO(sal-D-galacN)(H20)] and [V203(sal-D-glucN)(H20)], in which the ligands are Schiff bases derived from the condensation of salicylaldehyde (sal) and D(+)-Glucosamme (D-gcsN), D(+)-Galactosamine (D-galacN) and DGlucamine (D-glucN). A light-green complex formulated as [VO(pyr-D-glucN)(HeO)].X* was also prepared where pyr-D-glucN is the Schiff base derived from pyridoxal and D-glucamine, and X ÷ is Na ÷ or K*. The complexes were characterized by elemental analysis, IR, EPR, UV-VIS and Circular Dichroism spectroscopy. The preparation of complexes with the corresponding reduced Schiffbase complexes is also explored. We thank Fundo Europeu para o Desenvolvimento Regional and Funda~o para a Ci~ncia POCTI/35368/Qui/2000 and BD/18548/98 fellowship) for financial support.
e Tecnologia (project
Structural and spectroscopic characterization of new Cu(II)-dipeptide complexes Maria H. Torrea, Gianella Facchin a, Eduardo Kremera, Enrique J. Baranb, Oscar E. Piroc aQuimica Inorgdzniea, D.E.C., Facultad de Quimica, UDELAR, Gral. Flores 212, 11800, Montevideo, Uruguay, (e-mail [email protected]) bCEQUINOR; CDepartamento de Fisica and IFLP, Facultad de Ciencias Exactas, UNLP, I900, La P/ata, Argentina Cu(II) complexes containing peptides as ligands are often good model systems to obtain a better insight into the characteristics of naturally occurring copper proteins. As a part of our study of a series of copper compounds with peptides two new Cu(II) complexes of stoichiometry [Cu(L-dipeptide)] containing L-AIa-L-Ile and LAla-L-Thr were prepared and characterized by single crystal X-ray diffractometry, IR i~ e.4 S and electronic spectroscopy. Results were compared with those of Cu-L-Ala-L-Val and Cu-L-AIa-L-Phe previously studied, i In the four complexes the Cu(II) is at the square base center of an elongated pyramidal environment, equatorially coordinated by a dipeptide molecule acting as a tridentate ligand as shown in the figure. The equatorial copper coordination gives rise to polymeric structures in the lattice. Superoxide dismutase (SOD)-like activity was also tested. The values of ICs0, using the nitrobluetetrazoliurn/superoxide reduction assay were 3.3 E-4 M (CuL-Ala-L-Ile) and 9.9 E-5 M (CuL-Ala-L-Thr). 1.
G. Facchin, M. H. Torre, E. Kremer, E. J. Baran, O. Piro, Z. Naturforsch, 55b, 1157 - 1162 (2000)
Journal of Inorganic Biochemistry 86 (2001)
Studies
o n E u II
459
chelates in relation to magnetic esonance imaging
t~va T 6 t h , L f i s z l 6 B u r a i , A n d r 6 E. M e r b a c h
Institute of Inorganic and Analytical Chemistry, University of Lausanne, BCH, 1015 Lausanne, Switzerland (e-mail. Eva.Jakab- Toth@icma. unil. ch) TETA ODDM DOTA ODDA
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The recent interest in ~:he aqueous solution chemistry of Eu II chelates has been prompted by two main aspects. L First, Eu ~ is isoelectronic with Gd m, therefore studying relaxation phenomena on the Eu H analogues can give further insight into the mechanisms that govern relaxation on Gd III complexes as well and thus help the development of novel MRI contrast agents. On the other hand, one can take advantage of the redox abilities of Eu ~z and design EuH/Eu ~ based responsive MRI contrast agents which are sensitive to the redox state of the biological environment. We will present recent results related to these aspects. 2.
Toth E., Burai L. and Merbach A.E., Coord. Chem. Reviews, (2001) in press.
-0.6
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,
-02
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E vs AgZAgCI{V)
Fig. 1. The redox potential of Eull/Eu 111 changes substantially as a function of the ligand
Density functional description of the spin states involved in the early stages of the dioxygenation of [(MeC(CH2PPhz)3)Ir(cathecolate)]+: a possible model of dioxygenases Federico Tottia, Alessandro Bencinia, Andrea Scozzafavab "Dipartimento di Chimica, Universith di Firenze, Via Maragliano 75/77, 50144, Firenze ( I t a l y ) (e-mail. [email protected], i 0 ~Dipartimento di Chimica, Laboratorio di Bioinorganica, Universitgt di Firenze, Via Capponi 7, 50121, Firenze (Italy) The earlier part of' our work we have been applied to, has been devoted to the study o f the basic points related to the reaction path involving the [(triphos) M(cathecolate)] + + 02 (M = Co(III) and Ir(III)) moieties in their fundamental states. Nevertheless, the above reactions are spin-forbidden, and the spin-orbit coupling is supposed to be the most probable mechanism that can allow the reactions. In order to have an insight in such a topic, we have carried out calculations on the excited states for the already found stationary points, plus some others on different potential surfaces. In this framework we have used both pure density functional approach (VWN + Stoll correction) with slater-type basis sets (ADF, Amsterdam Density Functional package) and a hybrid one (B3LYP) with gaussian-type basis sets (Gaussian98 package). As regards the [(triphos)Ir(III)(cathecolate)]+ + 02 reaction, we have attempted to scan the potential surfaces for the low lying spin states possibly involved in the reaction, that is 3A state and the two possible ~A states. The importance of the mono-protonated cathecol binding in the extra-diol cleavage has been also investigated following the same computational protocol used for the intra-diol dioxygenases. In this case we have taken into consideration the [(triphos)Ir(III)(cathecolate)H]2+ + O2 reaction. Single points solvent calculations (IPCM) have been also performed for several geometries with Gaussian98. A new plausible mechanism for the intra- and extra-dioxygenases has been then suggested supported by the computed data obtained in this work.
460
Journal of Inorganic Biochemistry 86 (2001)
Dinuclear manganese complexes as structural and spectroscopic models for manganese catalases Michael U. Triller a, Vincent L. Pecoraro b, Annette Rompel a and Bernt Krebs a " Anorganisch-Chemisches Institut der Westffilischen Wilhelms- Universitgit, Wilhelm-Klemm-Str. 8. 48149 Miinster, Germany, b Department of Chemistry, University of Michigan, 930 North University Boulevard, Ann Arbor, Michigan 48109-1055, U,S.A. Manganese catalase from T. thermophilus (TTC) catalyzes the disproportionation of H202 to H20 and O2.1 Four oxidation states of the enzyme have been characterized, namely the catalytically active (2,2) and 2_ (3,3) states and the inactive (2,3) and (3,4) states. I The dinuclear complexes [Mn(L)(,u-OAc)]2[CIO4]2 1, [Mn2(L)2(/.t-O)(/.t-OAc)][ClO4]3 2 and [Mn(L)(,u-O)]2[C104]2[PF6] 3 (L = Bis(picolyl)((l-methylirnidazol-2-yl)methyl)amine) are structural and spectroscopic models for the (2,2), (3,3) and (3,4) states, respectively. Like the (2,2) form of TTC, a UV/Vis spectrum of 1 shows no absorption in the visible region, while 2_ exhibits a band at 495 nm (492 nm for the (3,3) form of TTC). I EPR measurements reveal a 16 line spectrum for 3. Complexes 1, 2 and 3 disproportionate H202. Attempts to synthesize model complexes for the chloride inhibited form of TTC yielded [Mn(L)(C1)2] [CIO]44 and [(Mn(L)(C1))2~-O)][C104]2 5_.-' 3. 4.
Whittaker M.M., Barynin V.V., Antonyuk S.V., Whittaker J.W., Biochemistry, 38, 9126 - 9136 (1999) Antonyuk S.V., Melik-Adamyan V.R., Popov A.N., Lamzin V.S., Hempstead P.D., Harrison P.M., Artymyuk P.J., Barynin V.V., Crystallography Reports, 45, 111-122 (2000)
pH dependent Hg(II) coordination in metallothioneins: a 199mHg TDPAC study W. Tr6gera, B. Ctorteckaa, P. Fallerb, M. Va~b.kb and
the I S O L D E Collaboration c
alnstitute of Experimental Physics II, University of Leipzig, Linn~strafle 5, 04103 Leipzig, Germany (email. troeger@physik, uni-leipzig.de) blnstitute of Biochemistry, University of Ziirich, Winterthurerstr. 190, 8057 Ziirich, Switzerland ~CERN 1211 Geneva. Switzerland 7 Mammalian metallothioneins (MT) are ubiquitous, 08 o,oo~ ~t cysteine-rich proteins of low molecular weight which bind dl 0 o,6 Hg'-MT •~.4 ii pH 8.6 metals ions such as Zn(II), Cd(II), Cu(I) and Hg(II) in metalthiolate clusters. They play an important role in the metabolism and in the modulation of the essential trace element zinc and copper and in the binding of toxic heavy ~6 ~ Hg'-MT metals. The latter suggests also the involvement in cellular ~os[" '1"7 -6.4! , pH 2 detoxification mechanisms. Several 3D structures have been 02~, I 'w' ~. • °'° - J v"~,'-,~,q',~h..,~.~ solved for mammalian Me(II)7-MT, containing Zn(II) and/or -o.o5 L. i Cd(II) ions. These metal ions are tetrahedrally coordinated by o 2 4 6 B 10 12 o 5 lO 15 20 both bridging and terminal thiolates in cluster structures. Here, Frequency [Grad/s] Time [ns] we report on Hg(II) coordination in rabbit liver MT by time Fig 1: TDPAC time spectra (left) and thei Fourier transforms differential perturbed angular correlation (TDPAC) (right) of Hg7-MT at pH 8.6 (top) and pH 2 (bottom). The low frequencyat pH 8.6 indicates a tetrahedral and the high frequency spectroscopy. With TDPAC the local environment of the at pH2 a diagonal Hg coordinationgeometry TDPAC probe, here 199rnHg, can be studied by the correlation of two subsequent gamma quanta emitted by the probe• The titration of apo-MT with Hg(II) (1-7 equiv.) revealed mainly two- and fourfold Hg(II) coordinations depending on Hg(II) concentration and pH. At pH 2.0 twofold coordinations dominate whereas at pH 8.6 also higher coordination numbers occur.
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