- Email: [email protected]
Abstracts Poster Presentations (T)
Journal of lnorganic Biochemistry 86 (2001)
311
The synthesis and complexing properties of monoesters of aminoalkylphosphonic acids P e t r a Lebdugkovfi. P e t r H e r m a n n , J a n K o t e k , P a v e l Vojti~ek and I v a n L u k e ~
Department of lnorganic Chemistry, Faculty of Science, CharlesUniversity,Hlavova 2030, Prague 2, Czech Republic ( e - m a i l : petraleb@natur, cuni.cz) The synthesis and complexing properties of two aminoalkylphosphonate monoesters are studied both in the solution and the solid state. Their properties are compared to known aminoalkylphosphonate diesters. The proton dissociation constants (pKa) and copper (II), cobalt (II), nickel (II) and zinc (II) complex stability constants of both ligands have been determined. The data reveals the following sequence for the proton dissociation constants: phosphinic acid < monoethyl alkylphosphonate < carboxylic acid < phosphonic acid O OH I /---X A ' O The pKa values of monoethyl alkylphosphonates are much EtO--P.~ .N N P. . . . closer to the pKa of the phosphinic acids than to the ones of OH O OH phosphonic acids. The stability constants obtained for the metal 1 2 complexes follow the same trend. The crystal structure of cobalt (II), nickel (II) and zinc (II) complexes of zwitterionic ligand (1), and copper (II) complex of zwittnerionic ligand (2) were determined. All the three complexes of the ligand (1) are isostructural, and the ligand coordinates with only the nitrogen atoms, forming an octahedral complex with four molecules of coordinated water. On the other hand, the copper (II) complex of ligand (2) is ligand-bridged and therefore polymeric. The co-ordination sphere of each copper atom is square pyramidal, the basal sites are occupied by two nitrogen and two oxygen atoms of two molecules of ligand (2); the apical site is occupied by a molecule of water.
Modeling catechol dioxygenases using benzimidazole-based ligands Hung Kay Lee, ChunPong Lam, Yee-LokWong Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, HONG KONG (e-mail: hklee@cuhk, edu.hk) Catechol dioxygenases belong to a family of non-heme iron enzymes which catalyze the oxidative cleavage of catechols. ~ Biomlmetic model complexes derived from a variety of supporting ligands have been reported as structural and functional mimics for the enzymes. 2 However, model complexes supported by imidazole- or benzimidazole-based ligands receive relatively less attention. Accordingly, we set out to prepare and characterize a series of iron(III) catecholate complexes containing ligands with benzimidazole functionality. Treatment of [N,N-bis(2-benzimidazoylmethyl)-N-(3,5-di-tert-butyl-2-hydroxybenzyl)]amine (LH) with Fe(CIO4)3, di-tert-butylcatechol (dbcH2), and triethylamine in methanol gave the corresponding Fe(III)-catecholate complex [Fe(L)(dbc)] (1) as purple crystals. Compound 1 has been characterized by elemental analysis, high resolution mass spectrometry, and EPR spectroscopy. The reactivity of 1 toward dioxygen has also been investigated. 1. Lipscomb, J. D.; Orville, A. M., Metallons Biol. Syst., 28, 243-298 (1992) 2. Que, L., Jr.; Ho, R. Y. N., Chem. Rev., 96, 2607-2624 (1996) This work was supported by a Direct Grant (A/C 2060127) of The Chinese University of Hong Kong.
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Journal of Inorganic Biochemistry 86 (2001)
E l e c t r o n transfer triggered folding of four-helix b u n d l e s Jennifer C. Lee, Michele A. McGuirl, Harry B. Gray, and Jay R. Winkler Beckman Institute, MC 139-74, California Institute of Technology, Pasadena, CA, 91125, USA (email: lee@caltech, edu) Cytochrome c' (cyt c'), a four-helix bundle from the photosynthetic bacterium Rhodopseudomonas palustris, has a high-spin, five-coordinate heme, axially ligated by HisUT; it has a reduction potential (100 mV) high enough to permit electron transfer (ET) triggered folding. Wild-type Fe(II)-cyt c' folding reveals very heterogeneous kinetics 1 2 1 1 spanning a wide range of time; there are fast-(-10 3 s-), intermediate-(10-10 s-), and slow-folding (10 -~ s-~) populations, with the latter two likely containing methionine-ligated (Met is or Met~5) ferrohemes. In the absence of misligation (Fe(II)-CO and Fe(III)-cyt c'), cyt c' requires 500-1000 ms to adopt a folded structure. The presence of an intermediate (-10 ~- s- ) folding phase in Fe(II)-cyt c' implies that normative methionine ligation can facilitate refolding. The gene for cyt c' has been cloned and expressed in E. coli. so that site-directed mutagenesis can be used for folding studies. Single and double Met-mutants (Metl5Leu and Met25Ala) have been prepared in order to probe the roles of misligated intermediates during folding.
let
His
let
117
JCL acknowledges the Ralph M. Parsons Foundation for a graduate fellowship. This work was supported by NSF and the Arnold and Mabel Beckman Foundation
E n z y m e electrokinetics: E n e r g e t i c s of succinate oxidation by E. coli F u m a r a t e R e d u c t a s e . Christophe L6ger, a Kerensa Heffron, a Harsh R. Pershad, b Elena Maklashina, c C6sar Luna-Chavez, d Gary Cecchini, c Brian A. C. Ackrell c and Fraser A. Armstrong. a Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, UK. (e-mail: [email protected]) b Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA c Molecular Biology Division, VA Medical Center and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94121, USA d Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA. Protein Film Voltammetry I is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory metalloflavoenzyme, the membrane extrinsic domain ofE. Coli fumarate reductase, 2 which is homologous to mitochondrial Complex II (succinate dehydrogenase). The oxidative activity of the enzyme as a function of the driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the two reduction potentials of the flavin (FAD), the stability of the semiquinone, relevant protonation states and pH-dependent succinate-enzyme dissociation constants for all three redox states of the FAD. The sequence of binding and protonation events over the whole catalytic cycle is deduced) 1. 2. 3.
Armstrong et al., Chem. Soc. Rev. 26(3) 169-179 (1997), Electrochim. Acta 45(15-16) 2623-2645 (2000). Iverson et al, Science 284(5422) 1961-1966 (1999), Ohnishi et al., Structure 8 R23-R32 (2000), Ackrell et al. FEBS Lett., 466 1-5 (2000). Leger et al., submitted to Biochemistry.
Journal of Inorganic Biochemistry 86 (2001)
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Molecular modeling of the three-dimensional structure of Fe(II)-bleomycin. Are the Co(II) and Fe(II) adducts isostructural? Teresa E. Lehmarm, a and Maria Luisa Serrano b "Laboratorio de Andlisis Instrumental, Centro de Quimica, Instituto Venezolano de lnvestigaciones Cientificas (IVIC), Caracas 1020-A, VENEZUELA. (e-mail." tlehmann@quimica,ivic.ve). bLaboratorio de Modelaje Molecular, Facultad de Farmacia, Universidad Central de Venezuela, Caracas 1041-A, VENEZUELA. The molecular modeling of Co(II)-bleomycin previously performed by us through NMR and molecular dynamics, indicates that the most favorable structure for this complex is six-coordinate, with the secondary amine in [3aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole tings, respectively, and the amide nitrogen in 13hydroxyhistidine as equatorial ligands.~ The primary amine and either the carbamoyl oxygen or a solvent molecule, are proposed to occupy the axial sites. In this report, the results of the molecular modeling of Fe(II)-bleomycin are presented. As found for the Co(II) adduct, the results of our investigation on ferrous-bleomycin, are most consistent with a six-coordinate structure with five endogenous N-donor and a solvent molecule or the carbamoyl oxygen as the sixth ligand. The proposed isostructurallity between the Co and Fe adducts of bleomycin was also addressed in the study described herein, Comparisons of our best Co(II)- and Fe(II)-bleomycin models with the NMR-generated structures for HOO-Co(III)-pepleomycin and HOO-Co(III)-bleomycin, and with the X-ray structures of some model complexes, favor the models with only endogenous ligands as the structure probably held in solution by both Fe(II)-and Co(II)-bleomycin. 1.
Lehmarm T, Serrano M, Que L. Biochemistry 39, 3886-3898 (2000).
The Consejo de Desan'ollo Cientifico y Humanistico (CDCH) and The Instituto Venezolano de Investigaciones Cientifica (IVIC) are acknowledged for financial support.
Activation of peroxide by high-spin and low-spin Fe(III) centers Nicolai Lehnert a, Raymond Y. N. Ho b, Lawrence Que, Jr. b, Kiyoshi Fujisawa c, Edward I. Solomon a " Department of Chemistry, Stanford University, Stanford, CA 94305, USA (e-mail. [email protected], edu) h Department of Chemistry, University of Minnesota,207 Pleasant St. SE, Minneapolis, MN 55455, USA c Department of Chemistry. University of Tsukuba, Tsukuba 305-8571, Japan Mononuclear non-heme iron enzymes show a varity of different oxidation and hydroxylation reactions of substrates 1. These systems include intra- and extradiol dioxygenases, pterin-dependent hydroxylases, c~-ketoglutarate dependent enzymes, lipoxygenases and Rieske-type dioxygenases. For a number of these enzymes, high-spin Fe(III)-alkyl- and -hydroperoxo complexes have been proposed to occur along the reaction coordinate. However, no such intermediate has yet been isolated. The only well characterized intermediate is activated bleomycin, which is the low-spin Fe(III)-hydroperoxo complex of the anti-cancer drug bleomycin. In this study, a number of corresponding high-spin and low-spin Fe(III)-OOR (R = H, tertButyl) model complexes has been investigated by m e a n s of UV-Vis, MCD and resonance Raman spectroscopies and density-functional calculations. The spectroscopic properties and the electronic structure of these systems has been defined and their reactivity has been evaluated as a function of the R group on peroxide and the spin state of iron. In addition, density functional calculations have been performed on simplified analogs of potential enzymatic intermediates and their reactivity has been explored in relation to the results from the model complex studies. 4.
Solomon E. I., Brunold T. C., Davis M. I., Kemsley J. N., Lee S.-K., Lehnert N., Neese F., Skulan A. J., Yang Y.-S., Zhou J., Chem. Rev., 100, 235-349 (2000)
314
Journal of Inorganic Biochemistry 86 (2001)
The "strict" anaerobe d. gigas contains a membrane-bound oxygen respiratory chain R. S. L e m o s a ; C. M. G o m e s " , M. S a n t a n a a, J. L e G a l l b, A. V. X a v i e r a, M. T e i x e i r a a alnstituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, APT 127, 2780-156 Oeiras, Portugal(e-mail: miguel@itqb, unl.pO
bDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30622, USA Desulfovibrio are sulfate-reducing bacteria described as strict anaerobic organisms since their discovery, more than 100 years ago. In the last decade, several reports have shown that these bacteria are quite resistant to oxygen j. Here we report that Desulfovibrio gigas contains a membrane bound respiratory chain able to fully reduce dioxygen to water, with oxygen consumption comparable to those of aerobic organisms. In particular two components of this respiratory chain were characterized: a canonical membrane-bound terminal oxygen reductase of the cytochrome bd family and a fumarate reductase that is also capable, like most enzymes from this family, to function efficiently as succinate dehydrogenase. Fumarate reductase, a ......... Fum quinone and cytochrome bd were co-reconstituted in liposomes and this system is capable of coupling succinate oxidation with oxygen reduction to water. This "strict anaerobic" bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and / ., anaerobes are much more complex than originally thought, o, .-~ 1. Cypionka IL Annu.Rev.Microbiol. 54, 827-480 (2000) 2. Lemos RS et al, FEBSletters, 24828, 1-4 (2001) 3. Lemos RS et al, Journal of Bioenergetics and Biomembranes., in press This work was supported by FCT-Portugal grants 36560/99 and 36558/99 to MT. R.S.Lemos is recipient of PRAXIS XXI grant BD / 19867 / 99
A strongly coupled diiron / radical state in ribonucleotide reductase mutant Y122H of E. coil studied by 57Fe- and tH / 2H- ENDOR F. L e n d z i a n a, M. K o l b e r g a, G. Bleifuss a, G. L a s s m a n n a, A. Grfislund b, and W. L u b i t z a ~Max- Volmer-Laboratorium fur Biophysikalische Chemie, Technische Universitat Berlin, PC14, Strasse des 17. Juni 135, 10623 Berlin (e-mail: lendzian@echo, chem.tu-berlin.de) bDepartment of Biophysics, Stockholm University, Arrhenius Laboratories. S-10691 Stockholm, Sweden Ribonucleotide reductase (RNR) catalyses the reduction of ribonucleotides, which is crucial for DNA synthesis. Subunit R2 of RNR carries in the acitve state an anti-ferromagnetically coupled ~t-oxo bridged di-ferric center (FemFem) and a stable, weakly coupled tyrosyl radical, Y122", which are both essential for the catalytic activity. The tyrosyl radical is generated from the FeHFen state in a reaction with molecular oxygen, which is similar to the oxygen activation reaction in other diiron oxygen enzymes, e. g. methane monooxygenase. Here we report on a new paramagnetic center observed in R2 mutant Y 1221-I, where tyrosine 122 is replaced by a histidine, inducing changes in the diiron ligand sphere. The high-field EPR and 57Fe-ENDOR results indicate that this center is a S=1/2 state consisting of FetUFem and a strongly coupled, probably ligated, radical ~. The radical is identified as phenylalanine by experiments on selectively phenylalanine-deuterated samples. 1. M. Kolberg, G. Bleifuss, S. P6tsch, A. Gr~islund, W. Lubitz, G. Lassmann, and F. Lendzian J. Am. Chem. Soc., 122, 9856 (2000) Work supported by Deutsche Forschungsgemeinschaft (La 751/3-1, Le 812/1-2)
R2-Y122H
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Journal of lnorganic Biochemistry 86 (2001)
315
Towards functional mimics of manganese superoxide dismutases Elizabeth A. Lewis a, John R. Lindsay Smith a, Paul H. Waltona, Hicham Khodr b, Robert C. Hiderb, Stephen J. Archibalda and Gerard M. Giblinc <'Department of Chemistry, University of York, Heslington, York YOlO 5DD, UK (email. [email protected]) hDepartment of Pharmacy, Franklin Wilkins Building, King's College London, 150 Stamford Street, London, SE1 8A W CMedicinal Chemistry, Glaxo Smith Kline Research and Development, Gunnels Wood Road, Stevenage, Herts., SGI 2NY An overproduction of superoxide has been implicated as the cause of many inflammatory processes, reperfusion injury and degenerative diseases. As a result, there is a great deal of current interest in the design of functional models of superoxide dismutases (SODs), enzymes which play an important role in protecting cells through catalysis of superoxide disproportlonatlon. R~N--U'2----7"-. N/~R This work involves the condensation and subsequent reduction of H /~"'/ H substituted salicylaldehydes with cis,cis-l,3,5-triaminocyclohexane (tach) to NH 1 HI_ ~ form a series of tri-amine ligands (1) and their corresponding Mn(III) complexes. Systematic variation of the substituent, X, provides a mechanism R R= for tuning both the solubility and electronic properties of the complexes. 2 The relationship between the X substituent, metal-based redox behaviour, Mn(III) X=H,CH30,CH3,CI,NO2,N(CH3)3 +,SO3" X binding and SOD activities of these systems has been investigated with the ultimate goal of designing an effective SOD mimic. !. 2.
Riley D., Chem Rev., 99, 2573-2588, (1999) Lewis E. A., Lindsay Smith J. R., Walton P. H., Archibald S. J., Foxon S. and Giblin G. M., J. Chem. Soc., Dalton Trans., 1159-1161 (2001)
Six-coordinate and five-coordinate Fen(CN)2(CO)x-thiolate complexes (x = 1, 2): synthetic advances for iron site of [NiFe] hydrogenases Wen-Feng Liaw, Jiun-Hung Lee, Hung-Bin Gau, Chien-Hong Chen, Shiou-Ju Jung, Wen-Yuan Chen, Ching-Han Hu, Chen-Hsiung Hung. Department of Chemistry, National Changhua University of Education, Changhua, 50058, Taiwan ([email protected]) The dicyano-dicarbonyl iron(II)-thiolate complexes trans, cis-[(CN),(CO)2Fe(S, S-C-R)]- (R = OEt (2), N(Et)2 (3)) were prepared from reaction of [Na][S-C(S)-R] and [Fe(CN)2(CO)3Br]- (1) obtained from oxidative addition of cyanogens bromide to [Fe(CO)4(CN)] respectively. Photolysis of THF solution of complexes 2 and 3 under CO atmosphere led to formation of the coordinate-unsaturated iron(II)-dicyanocarbonyl thiolate compounds [(CN)2(CO)2Fe(S , S-C-R)] (R = OEt (4), N(Et)2 (5)) respectively, the potential iron-site structural and functional model compound of active site of [NiFe] hydrogenases. The IR (CN) stretching frequencies and patterns of complexes 4 and 5 unambiguously identified two CN- ligands occupying cis positions. The theoretical calculations suggest that the architecture of five-coordinate complexes 4 and 5 with a vacant site trans to the CO ligand and two C N ligands occupying cis positions serves as a conformational preference. Complexes 2 and 3 were reobtained when THF solutxon of complexes 4 and 5 were exposed to CO atmosphere at ambient temperature individually. Isotopic shift experiments demonstrate the lability of carbonyl ligands of complexes 2, 3, 4 and 5. The facile formation of five-coordinate complexes 4 and 5 implicate that the strong mdonor, weak n-acceptor CN- ligands play a key role in creating/stabilizing five-coordinate iron(lI) complexes 4 and 5 with a vacant site trans to the CO ligand.
316
Journal of lnorganic Biochemistry 86 (2001)
Characterization of the particulate form of methane monooxygenase ( p M M O ) from methylococcus capsulatus (Bath) and methylosinus albus BG8 Raquel L. Lieberman, Peter E. Doan, Brian M. Hoffman, Amy C. Rosenzweig Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, IL 60208, U.S.A. ([email protected]) The conversion of methane to methanol is achieved in Nature by methane monooxygenase (MMO), an enzyme found in methanotrophic bacteria. This transformation is amazing because the enzyme selectively oxidizes the -100 kcal/mol C-H bond in methane at ambient temperature and pressure. All methanotrophs utilize the particulate form (pMMO), whereas some can also express the well-characterized soluble form (sMMO). It is currently believed that the active site o f p M M O contains copper and possibly iron, but the details of the nuclearity of the metal cluster, the identity and arrangement of the ligands, and the electronic environment have not been well established. We have purified pMMO from M. capsulatus (Bath) to homogeneity. This methanotroph expresses both the soluble and particulate form of methane mofiooxygenase. We are also purifying pMMO from M. albus BG8, a methanotroph that expresses only the particulate form. The pMMO enzyme from M. capsulatus (Bath) contains three subunits of molecular masses 29, 45, and 31 kDa in a stoichiometry of c~213:'{2. Reverse-phase HPLC data show that the [3 subunit is the least hydrophobic, followed by y and c~, respectively. We measure up to six copper ions per 200 kDa enzyme in our purified samples. All of the copper ions are EPR silent, suggesting that they are present as Cu(I).
Structural studies on desulfovibrio desulfuricans A T C C 27774 multiheme nitrite reductase - characterization of the subunits L. M. Lima Gongalves a,b, C. Cunha a, G. Almeida a, S. Macieira a, C. Costa a, j. Lampreia a, M. J. Romeo a, j. j. G. Moura a, I. Moura a a Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de CiFncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte de Caparica, Portuga.l oInstituto Superior de Cidncias da Sa~de-Sul, Campus Universitdrio - Quinta da Granja, 2825-511 Caparica, Portugal (e-mail: [email protected]) Multiheme cytochrome c nitrite reductase (NiR) isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 is a membrane bound enzyme that catalyses the dissimilatory nitrite reduction to ammonia in a six-electron step. Initially the enzyme was described as a 66kDa monomeric protein, containing six e type hemes as prosthetic groups. 1 Later on, it was shown that NiR is a hetero-oligomeric complex composed by two subunits (63kDa and 19kDa) both containing c-type hemes z- the large subunit contains 5 heroes and the small most probably 4 hemes. The primary sequence of the two subunits are presented, discussed and compared with the D. vulgaris genomic data. We also report biochemical and spectroscopic studies on the two isolated subunits. So far, two 3D structures of the 5-heine containing large subunit o f NiR are available.4'5 The structure ofD. desulfuricans NiR was recently solved and.refined at 2.30 ~,. 3 1- Liu, M.-C., Costa, C. and Moura, I. Meth. Enz. 243,303-319 (1994). 2- Almeida, G. etal. J. Inorg. Biochem. 74, 63 (1999). 3- Dias, J.M. et al. Acta Crystallographica D56, 215-217 (2000). (preliminary results) 4- Einsle, O. et al. Nature 400, 476-480 (1999). 5- Einsle, O. et a.l J.Biol. Chem. 275, 39608-39616 (2000). We acknowledge the Fundaq~o para a Ci~ncia e a Tecnologia for the financial support.
Journal of Inorganic Biochemistry 86 (2001)
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Model complexes to mimic the 7t-stacking interaction in galactose oxidase X i a o m i n g L i u a n d M a l c o l m A. H a l c r o w *
School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK, (e-mail." x. [email protected], uk) Galactose oxidase (GOase) is a type II copper enzyme from wood-rot fungi that catalyses the oxidation of primary alcohols to aldehydes by molecular oxygen] An oxidisable tyrosine residue in GOase enables this enzyme to catalyse a two-electron oxidation reaction. The single crystal X-ray structure shows a square-pyramidal centre with two phenoxide donors (Figure 1). 2 The basal tyrosinate ligand is chemically modified with an ortho-thioether linkage formed by an oxidative cross-linking Tyr495 reaction to a cysteine side-chain, and is involved in a n-stacking interaction with a neighbouring indole ring. To further understand how the structural features of the GOase radical contribute to its chemistry, 3 we have synthesised new phenols based on the dibenzo[c,h]bicyclo-[4.4.1]undeca-3,8-dien-ll-one skeleton. 4 Acetalation of the ketone group in these compounds causes a conformational change, from an open boat-chair to a closed form in which the two annelated His496 Acetate Cys228 rings overlie each other. Comparison of the spectroscopic, structural and redox chemistries of the four derivatives in Figure 2 and their complexes has allowed Figure 1. Crystal structure of the us to understand in some detail how a thioether substituent and rt-rt interaction each contribute to the properties of a phenoxyl radical. GOase active site. 2
1.
Kaim W. and Rall J., Angew Chem., Int. Ed.
O
n1343601996 2. Ito N., et al, Nature, 350, 87-90 (1991). 3~ Halcrow M.A., et al, J. Chem. Soc., Dalton Trans., 4025-4036 (1997). 4, Mataka S., et al, Synlett., 1211-1227 (2000). We thank EPSRC (XL) and the Royal Society (London, MAH) for financial support.
Antitumor
HO~ O H
OH R = H, SMe OH F i g u r e 2. N e w p h e n o l l i g a n d s p r e p a r e d in this
platinum complexes bend adjacent purines with monofunctional binding
trans
Y a n g z h o n g L i u a, M a r i a F. S i v o b, G i o v a n n i N a t i l e b, a n d E i n a r S l e t t e n a
"Department of Chemistry, University of Bergen, Allegt. 41, N-5007 Bergen, Norway (e-mail. einar.sletten@kj, uib.no) bDepartment of Pharmaceutical Chemistry, University of Bari, Via E. Orabona 4, 1-70125,.Bari Italy. The new antitumor drug trans-EE binds to DNA forming essentially monofunctional adducts which appear to be responsible for the anticancer activity. We have previously shown that a DNA oligomer can be bent 45 degree towards the minor groove by trans-EE mono-binding 1, such a binding is comparable, in size, to that given by cisplatin which chelates on adjacent purines and bends DNA towards the major groove. A question that has arisen from this investigation, is how this mono-binding could bend DNA to such an extent? f Three monofunctional adducts of trans-EE with the nucleotide dimers, rApG, dApG and -~"~ . dGpA were synthesized. The kinetic processes were monitored by HPLC and adduct structures were studied by NMR spectroscopy. Guanine N7 was the only binding site in each adduct and in all of them a large down field shift of the G H8 signal was observed. The models were built using Insight II and were subsequently subject to energy minimization and molecular dynamics (BIOSYM) calculations using distance constraints extracted from the ROESY maps. The structure shows that the adenine base is bent 34 degree away from its initial geometry in the rApG adduct. The bending is probably caused by the hydrogen bond between a trans-EE imino rApGadduct proton and the adenine N7. 3.1 A between two nitrogen is suitable to form this hydrogen binding. The conversion to bifunctional adducts was also studied. 1. Andersen B., Margiotta, N., Coluccia M. Natile G. and Sletten E. Metal-Based Drags 7, 23-32 (2000)
318
Journal of Inorganic Biochemistry 86 (2001)
First examples of luminescent cyclometallated iridium(III) complexes as labelling reagents for biological substrates Kenneth Kam-Wing Lo,* Dominic Chun-Ming Ng and Chi-Keung Chung Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, KowIoon, Hong Kong, P.R. China We report the syntheses, photophysical and electrochemical properties of two novel cyclometallated iridium(III) complexes [Ir(ppy)2(phen-NCS)](PF6) (1) and [Ir(ppy)z(phen-NHCOCHzI)](PF6) (2) (Hppy = 2-phenylpyridine, phenNCS = 5-isothiocyanato- 1,10-phenanthroline, phen-NHCOCHzI - 5-iodoacetamido- 1,10-phenanthroline). These two iridium(III) complexes exhibit intense and long-lived orange-yellow luminescence upon photoexcitation in CH3CN at 298 K (1: ~-em = 608 nm, "Co = 0.41 p.s; 2: ~-em : 590 rim, Zo = 0.59 Its). The complexes show reversible metal-based oxidation couples at ca. +1.26 V vs. S.C.E. in CH3CN at 298 K. The first irreversible reduction waves of 1 and 2 occur at ca. - i . 2 1 and -1.08 V, respectively. The emission of the complexes in room-temperature fluid solutions is assigned to originate from a metal-to-ligand charge-transfer [dT~(Ir) --~ 7z*(diimme)] triplet excited state. Since the isothiocyanate and iodoacetamide groups can react with the primary amine and sulfhydryl groups, respectively, 1 and 2 have been used to label a universal M13 reverse sequencing primer modified with an amine or a sulfhydryl group at the 5'-end. The photophysical and hybridisation properties of the labelled oligonucleotides have been investigated. In addition, these two complexes have also been coupled to human serum albumin. The bioconjugates 1-HSA and 2-HSA display intense and long-lived emission at Xcm = 562 nm ['tl - 0.90 ~ts (33 %), "~2= 0.15 Its (67 %)] and ~em : 570 nm [rl = 0.66 ~ts (19 %), z2 0.07 Its (81%)], respectively, in degassed 50 mM Tris-C1 pH 7.4 at 298 K. In the presence of oxygen, the emission lifetimes exhibit only a small decrease, suggesting that the labels are shielded in the interior of the protein matrix. We acknowledge financial support from the Hong Kong Research Grants Council and the City University ofHong Kong.
An artificial hemoprotein: m i m o c h r o m e IV Angela Lombardia, Flavia Nastria, Omella Maglio a, Daniela Marascoa, Massimiliano Colettab, Vincenzo Pavonea Department of Chemistry, University of Naples "Federico II", via Cynthia 45, 1-80126 Naples, [TALY (e-mail: [email protected]) b Department Experimental Medicine and Biochemical Sciences, University ofRoma Tor Vergata, Via di Tor Vergata 135, 00133 Roma Peptide-based metalloprotein models have recently emerged as an efficient tool for reproducing the active site properties and reactivity. We have applied this approach to the development of a new class of ~ A ' ~ hemoprotein mimetics, named mimochromes ~. They are C2 simmetric dimers made up by a deuteroporphyrin ring covalently linked to two helical peptide chains; the heme iron is axially ligated by one or two His residues. The prototype, mimochrome I, contains two nonapeptides, which represent the smallest sequence able to fold into a-helical conformation and to cover the heine plane. The structural characterization of mimochrome I iron and cobalt complexes confirmed the design, even though it revealed some unexpected features. The peptide chains were able to fold up around the porphyrin in two different fashions, giving rise to A and A diastereomers. In order to drive the folding of mimochromes into a unique diastereomer, we further refined the structure by design. One approach was the substitution of amino acids in key positions, which may provide intra-molecular inter-helical interactions and stabilize the sandwich. This approach led to mimochrome IV, which was fully characterized in both the apo- and metal-forms. All the experimental results indicate a unique topology ofpeptide chains, as designed. 1.
Nastri F., Lombardi A., D'Andrea L., Sanseverino M., Maglio O. and Pavone V., Biopolymers, 47, 5-22 (1998)
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
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Miniaturized metallo proteins: coordination properties of METP2 A n g e l a L o m b a r d i a, F l a v i a N a s t r i a, D a n i e l a M a r a s c o a, O m e l l a M a g l i o a, E n z o L a u r e n t i b, R o s a Pia Ferrari b, V i n c e n z o P a v o n e a
"Department of Chemistry, University of Naples "Federico II', via Cynthia 45. 1-80126 Naples, /TAL Y (e-mail. iombardi@chemistry, unina.it) b D e p a r t m e n t o f C h e m i s t r y I . F . M . , U n i v e r s i t y o f T o r i n o , 1-10125 T o r i n o , I t a l y The search for model systems able to reproduce the properties of natural metalloenzymes is one of the most pursued fields in bioinorganic chemistry. Synthetic metal complexes can be useful to provide information on the mechanism of action of natural systems. By changing the nature of the ligand, is possible to explore and rationalize the factors that tune the properties of the metal, in a way that could be easier respect to the use of mutant proteins. With the aim of obtaining peptide-based metalloprotein mimetics, we have recently employed a miniaturization process in the design of peptides, able to bind metal ions in a tetrahedral geometry. We developed a suitable prototype, METP (Miniaturized Electron Transfer Protein), which was designed using the mbredoxin as template structure. METP contains a Cys4 metal binding site and is made up of two identical undecapeptides self assembled upon metal ion coordination. It was shown to bind, in a tetrahedral geometry, Co(II), Fe(II)/Fe(III), Zn(II), as expected 2. With the aim of construct different tetra-coordinated metal-binding sites, we synthesized new molecules, which differ from the prototype METP for the nature of the coordinating residues. METP2 encloses a N4 metal binding site; its coordination properties, as ascertained by several spectroscopic techniques, will be presented. i. 2.
DeGrado W.F., Summa C.M., Pavone V., Nastri F., Lombardi A., Annu. Rev. Biochem. 68, 779-819(1999). Lombardi A., Marasco D., Maglio,O., Di Costanzo L., Nastri F., Pavone V. Proc. Nat. Acad. Sci. USA 97, 1192211927 (2000)
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
The effect of polyamines (PA) on the coordination mode in Cu(II) complexes with adenosine 5'-triphosphate (ATP) and cytidine 5'-triphosphate (CTP) Lechoslaw Lomozik,Anna Gasowska Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland (e-mail. [email protected]) A study of the mode of Cu(II) coordianation with polyammes was performed for the systems Cu(II)/ATP(or CTP)/PA; (PA = putrescine (Put), 1,3-diammopropane (tn), spermidine (Spd), 3,7-diamino-4-azaheptane (3,3-tri), spermine (Spm) and 1,11-diamino-4,8-diazaundekane (3,3,3-tet)). The results proved the formation of heteroligand molecular compounds, protonated complexes and CULL' type complexes, each with a different type of metallation being a result of the interaction with polyamines. In the parent complexes Cu(ATP) and Cu(CTP), the copper(II) ions are coordinated through the oxygen atoms of the phosphate groups and the nitrogen atoms of the purine or pyrimidine rings ({N,O} chromophore). In the mixed complexes, the interactions of the polyamine with the anchoring complexes Cu(ATP) o r Cu(CTP) lead to an increase in the coordination efficiency of the phosphate groups of the nucleotides. In molecular complexes, e.g. Cu(ATP)H3(Spd) and Cu(CTP)H2(Put), the fully protonated polyamine is involved in noncovalent interactions with the donor centres of the purine or pyrimidine rings and it is located outside the inner coordination sphere. The metallation occurs through oxygen atoms of the phosphate groups (and in the case of ATP through the donor atom N(7) as well). In the protonated species, e.g. Cu(ATP)H2(Spd) and Cu(CTP)H3(Spm), the copper ions are coordinated by the oxygen atoms of the phosphate groups and deprotonated nitrogen atoms of the polyamine ({N×,O} chromophore). The complex is additionally stabilised as a result of intramolecular non-covalent interactions of the protonated amine groups of the polyamine with the donor centres of the nucleotide. It has been found that in the mixed-ligand CULL' type complexes, e.g. Cu(ATP)(3,3-tri) and Cu(CTP)(3,3,3-tet), the inner coordination sphere of the central atom comprises the oxygen atoms of the phosphate group from A T P o r CTP and the nitrogen atoms of the polyamine, whereas the donor nitrogen atoms from the nucleotide remain in the outer coordination sphere. Results of the studies of the metallation and non-covalent interactions in the model systems point to significant differences in the properties of the polyamines studied, which explains a higher biological activity of putrescine, spermidine and spermine compared to their homologues. The Polish State Committee for Scientific Research (KBN) is acknowledged for financial support.
Journal of lnorganic Biochemistry 86 (2001)
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Energy tranduction performed by a monomeric tetrahaem cytochrome Ricardo O. Louro a. Teresa Catarino a'b, Jean LeGall c, David L. Turner a'd, Ant6nio, V. Xavier a a lnstituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6, Apt. 127. 2780 Oeiras, Portugal. Fax. +351-21-4468766.(e-mail: [email protected] b Departamento de Quimica da Faculdade de Ciencias e Tecnologia da Universidade Nova de Lisboa, Monte da Caparica, Portugal c Department of Biochemistry and Molecular Biology, Universityof Georgia, Athens GA 30602. USA d Department of Chemistry, University of Southampton, Southampton S017 1Bd, UK. In order to understand the mechanisms of biological energy transduction, it is essential to determine the microscopic thermodynamic parameters which describe the properties of each of the various centres involved in the reactions, as well as their interactions. These interactions between centres can then be interpreted in the light of structural features of the proteins. Redox titrations of cytochrome c3 from Desulfovibrio desulfuricans ATCC27774 followed by NMR and visible spectroscopy were analysed using an equilibrium thermodynamic model. This characterisation allows the identification of several pairs of centres for which there are clear conformational (non-Coulombic) contributions to their coupling energies, thus establishing the existence of localised redox- and acid-base-linked structural modifications in the protein (mechano-chemical coupling) ~. The modulation of interactions between centres observed for this cytochrome favours a two-electron transition coupled with the acid-base transition which is important for the physiological function of this protein. These physiologically relevant coupling patterns are not easy to engineer by mutagenesis even when using structurally homologous proteins 2. I. Louro, R.O., Catarino, T., LeGall, J., Turner D.L.,and Xavier, A.V. submitted 2. Salgueiro, C.A., da Costa, P.N., Turner, D.L. Messias, A.C., van Dongen, W.M.A.M., Saraiva, L.M. and Xavier, A.V. Biochemistry in press (2001) The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Designing metalloenzymes containing heteronuclear metal assembly Yi Lu,.Jeffrey A.
Sigman, Alan Gengenbach, Xiaotang Wang, Thomas D. Pfister, Hyeon K. Kim
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA (e-mail: [email protected]) Recent studies have uncovered increasing number of metalloenzymes containing heteronuclear metal assembly, such as the heme-manganese center in manganese peroxidase (MnP) and heine-copper center in heme-copper oxidase (HCO). MnP is a heme peroxidase that is involved in biodegradation of lignin and bioremediation of aromatic pollutants, such as polychlorinated biphenyls. HCOs are a superfamily of terminal oxidases in the respiratory chains. Compared with homonuclear metal centers, these heteronuclear metalloenzymes are less well-understood due to their inherent complexity that results from the multiple metal ions. They also pose significant challenges for protein design. We have been able to use stable, easy-to~¢¢ ','" :~i \ produce, and well-characterized proteins (such as cytochrome c peroxidase -~ ~ V : r C;- -" (CcP) and myoglobin (Mb)) as scaffolds for making biomimetic models of MnP ~ and HCOf Using these simple protein models, we are learning the principle of how these metal-binding sites are constructed to perform their functions and how they can be t-me tuned to realize different structural and functional properties. Latest results from these projects will be presented and their implication related to the structure and function of MnP and HCO will be discussed.
HCO (thin) and CusMb (thick) 1.
B.K.-S. Yeung et al. Chem. & Biol. 4, 215 (1997); b) A. Gengenbach et al. Biochemistry, 38, 11425 (1999). 2. J.A. Sigman et al. J. Am. Chem. Soc. 121, 8949 (1999); b) J. A. Sigman et al. J. Am. Chem. Soc., 122, 8192 (2000). We thank the National Institute of Health of the United States of America for Financial support.
311
The synthesis and complexing properties of monoesters of aminoalkylphosphonic acids P e t r a Lebdugkovfi. P e t r H e r m a n n , J a n K o t e k , P a v e l Vojti~ek and I v a n L u k e ~
Department of lnorganic Chemistry, Faculty of Science, CharlesUniversity,Hlavova 2030, Prague 2, Czech Republic ( e - m a i l : petraleb@natur, cuni.cz) The synthesis and complexing properties of two aminoalkylphosphonate monoesters are studied both in the solution and the solid state. Their properties are compared to known aminoalkylphosphonate diesters. The proton dissociation constants (pKa) and copper (II), cobalt (II), nickel (II) and zinc (II) complex stability constants of both ligands have been determined. The data reveals the following sequence for the proton dissociation constants: phosphinic acid < monoethyl alkylphosphonate < carboxylic acid < phosphonic acid O OH I /---X A ' O The pKa values of monoethyl alkylphosphonates are much EtO--P.~ .N N P. . . . closer to the pKa of the phosphinic acids than to the ones of OH O OH phosphonic acids. The stability constants obtained for the metal 1 2 complexes follow the same trend. The crystal structure of cobalt (II), nickel (II) and zinc (II) complexes of zwitterionic ligand (1), and copper (II) complex of zwittnerionic ligand (2) were determined. All the three complexes of the ligand (1) are isostructural, and the ligand coordinates with only the nitrogen atoms, forming an octahedral complex with four molecules of coordinated water. On the other hand, the copper (II) complex of ligand (2) is ligand-bridged and therefore polymeric. The co-ordination sphere of each copper atom is square pyramidal, the basal sites are occupied by two nitrogen and two oxygen atoms of two molecules of ligand (2); the apical site is occupied by a molecule of water.
Modeling catechol dioxygenases using benzimidazole-based ligands Hung Kay Lee, ChunPong Lam, Yee-LokWong Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, HONG KONG (e-mail: hklee@cuhk, edu.hk) Catechol dioxygenases belong to a family of non-heme iron enzymes which catalyze the oxidative cleavage of catechols. ~ Biomlmetic model complexes derived from a variety of supporting ligands have been reported as structural and functional mimics for the enzymes. 2 However, model complexes supported by imidazole- or benzimidazole-based ligands receive relatively less attention. Accordingly, we set out to prepare and characterize a series of iron(III) catecholate complexes containing ligands with benzimidazole functionality. Treatment of [N,N-bis(2-benzimidazoylmethyl)-N-(3,5-di-tert-butyl-2-hydroxybenzyl)]amine (LH) with Fe(CIO4)3, di-tert-butylcatechol (dbcH2), and triethylamine in methanol gave the corresponding Fe(III)-catecholate complex [Fe(L)(dbc)] (1) as purple crystals. Compound 1 has been characterized by elemental analysis, high resolution mass spectrometry, and EPR spectroscopy. The reactivity of 1 toward dioxygen has also been investigated. 1. Lipscomb, J. D.; Orville, A. M., Metallons Biol. Syst., 28, 243-298 (1992) 2. Que, L., Jr.; Ho, R. Y. N., Chem. Rev., 96, 2607-2624 (1996) This work was supported by a Direct Grant (A/C 2060127) of The Chinese University of Hong Kong.
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Journal of Inorganic Biochemistry 86 (2001)
E l e c t r o n transfer triggered folding of four-helix b u n d l e s Jennifer C. Lee, Michele A. McGuirl, Harry B. Gray, and Jay R. Winkler Beckman Institute, MC 139-74, California Institute of Technology, Pasadena, CA, 91125, USA (email: lee@caltech, edu) Cytochrome c' (cyt c'), a four-helix bundle from the photosynthetic bacterium Rhodopseudomonas palustris, has a high-spin, five-coordinate heme, axially ligated by HisUT; it has a reduction potential (100 mV) high enough to permit electron transfer (ET) triggered folding. Wild-type Fe(II)-cyt c' folding reveals very heterogeneous kinetics 1 2 1 1 spanning a wide range of time; there are fast-(-10 3 s-), intermediate-(10-10 s-), and slow-folding (10 -~ s-~) populations, with the latter two likely containing methionine-ligated (Met is or Met~5) ferrohemes. In the absence of misligation (Fe(II)-CO and Fe(III)-cyt c'), cyt c' requires 500-1000 ms to adopt a folded structure. The presence of an intermediate (-10 ~- s- ) folding phase in Fe(II)-cyt c' implies that normative methionine ligation can facilitate refolding. The gene for cyt c' has been cloned and expressed in E. coli. so that site-directed mutagenesis can be used for folding studies. Single and double Met-mutants (Metl5Leu and Met25Ala) have been prepared in order to probe the roles of misligated intermediates during folding.
let
His
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117
JCL acknowledges the Ralph M. Parsons Foundation for a graduate fellowship. This work was supported by NSF and the Arnold and Mabel Beckman Foundation
E n z y m e electrokinetics: E n e r g e t i c s of succinate oxidation by E. coli F u m a r a t e R e d u c t a s e . Christophe L6ger, a Kerensa Heffron, a Harsh R. Pershad, b Elena Maklashina, c C6sar Luna-Chavez, d Gary Cecchini, c Brian A. C. Ackrell c and Fraser A. Armstrong. a Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, UK. (e-mail: [email protected]) b Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA c Molecular Biology Division, VA Medical Center and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94121, USA d Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA. Protein Film Voltammetry I is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory metalloflavoenzyme, the membrane extrinsic domain ofE. Coli fumarate reductase, 2 which is homologous to mitochondrial Complex II (succinate dehydrogenase). The oxidative activity of the enzyme as a function of the driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the two reduction potentials of the flavin (FAD), the stability of the semiquinone, relevant protonation states and pH-dependent succinate-enzyme dissociation constants for all three redox states of the FAD. The sequence of binding and protonation events over the whole catalytic cycle is deduced) 1. 2. 3.
Armstrong et al., Chem. Soc. Rev. 26(3) 169-179 (1997), Electrochim. Acta 45(15-16) 2623-2645 (2000). Iverson et al, Science 284(5422) 1961-1966 (1999), Ohnishi et al., Structure 8 R23-R32 (2000), Ackrell et al. FEBS Lett., 466 1-5 (2000). Leger et al., submitted to Biochemistry.
Journal of Inorganic Biochemistry 86 (2001)
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Molecular modeling of the three-dimensional structure of Fe(II)-bleomycin. Are the Co(II) and Fe(II) adducts isostructural? Teresa E. Lehmarm, a and Maria Luisa Serrano b "Laboratorio de Andlisis Instrumental, Centro de Quimica, Instituto Venezolano de lnvestigaciones Cientificas (IVIC), Caracas 1020-A, VENEZUELA. (e-mail." tlehmann@quimica,ivic.ve). bLaboratorio de Modelaje Molecular, Facultad de Farmacia, Universidad Central de Venezuela, Caracas 1041-A, VENEZUELA. The molecular modeling of Co(II)-bleomycin previously performed by us through NMR and molecular dynamics, indicates that the most favorable structure for this complex is six-coordinate, with the secondary amine in [3aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole tings, respectively, and the amide nitrogen in 13hydroxyhistidine as equatorial ligands.~ The primary amine and either the carbamoyl oxygen or a solvent molecule, are proposed to occupy the axial sites. In this report, the results of the molecular modeling of Fe(II)-bleomycin are presented. As found for the Co(II) adduct, the results of our investigation on ferrous-bleomycin, are most consistent with a six-coordinate structure with five endogenous N-donor and a solvent molecule or the carbamoyl oxygen as the sixth ligand. The proposed isostructurallity between the Co and Fe adducts of bleomycin was also addressed in the study described herein, Comparisons of our best Co(II)- and Fe(II)-bleomycin models with the NMR-generated structures for HOO-Co(III)-pepleomycin and HOO-Co(III)-bleomycin, and with the X-ray structures of some model complexes, favor the models with only endogenous ligands as the structure probably held in solution by both Fe(II)-and Co(II)-bleomycin. 1.
Lehmarm T, Serrano M, Que L. Biochemistry 39, 3886-3898 (2000).
The Consejo de Desan'ollo Cientifico y Humanistico (CDCH) and The Instituto Venezolano de Investigaciones Cientifica (IVIC) are acknowledged for financial support.
Activation of peroxide by high-spin and low-spin Fe(III) centers Nicolai Lehnert a, Raymond Y. N. Ho b, Lawrence Que, Jr. b, Kiyoshi Fujisawa c, Edward I. Solomon a " Department of Chemistry, Stanford University, Stanford, CA 94305, USA (e-mail. [email protected], edu) h Department of Chemistry, University of Minnesota,207 Pleasant St. SE, Minneapolis, MN 55455, USA c Department of Chemistry. University of Tsukuba, Tsukuba 305-8571, Japan Mononuclear non-heme iron enzymes show a varity of different oxidation and hydroxylation reactions of substrates 1. These systems include intra- and extradiol dioxygenases, pterin-dependent hydroxylases, c~-ketoglutarate dependent enzymes, lipoxygenases and Rieske-type dioxygenases. For a number of these enzymes, high-spin Fe(III)-alkyl- and -hydroperoxo complexes have been proposed to occur along the reaction coordinate. However, no such intermediate has yet been isolated. The only well characterized intermediate is activated bleomycin, which is the low-spin Fe(III)-hydroperoxo complex of the anti-cancer drug bleomycin. In this study, a number of corresponding high-spin and low-spin Fe(III)-OOR (R = H, tertButyl) model complexes has been investigated by m e a n s of UV-Vis, MCD and resonance Raman spectroscopies and density-functional calculations. The spectroscopic properties and the electronic structure of these systems has been defined and their reactivity has been evaluated as a function of the R group on peroxide and the spin state of iron. In addition, density functional calculations have been performed on simplified analogs of potential enzymatic intermediates and their reactivity has been explored in relation to the results from the model complex studies. 4.
Solomon E. I., Brunold T. C., Davis M. I., Kemsley J. N., Lee S.-K., Lehnert N., Neese F., Skulan A. J., Yang Y.-S., Zhou J., Chem. Rev., 100, 235-349 (2000)
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Journal of Inorganic Biochemistry 86 (2001)
The "strict" anaerobe d. gigas contains a membrane-bound oxygen respiratory chain R. S. L e m o s a ; C. M. G o m e s " , M. S a n t a n a a, J. L e G a l l b, A. V. X a v i e r a, M. T e i x e i r a a alnstituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, APT 127, 2780-156 Oeiras, Portugal(e-mail: miguel@itqb, unl.pO
bDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30622, USA Desulfovibrio are sulfate-reducing bacteria described as strict anaerobic organisms since their discovery, more than 100 years ago. In the last decade, several reports have shown that these bacteria are quite resistant to oxygen j. Here we report that Desulfovibrio gigas contains a membrane bound respiratory chain able to fully reduce dioxygen to water, with oxygen consumption comparable to those of aerobic organisms. In particular two components of this respiratory chain were characterized: a canonical membrane-bound terminal oxygen reductase of the cytochrome bd family and a fumarate reductase that is also capable, like most enzymes from this family, to function efficiently as succinate dehydrogenase. Fumarate reductase, a ......... Fum quinone and cytochrome bd were co-reconstituted in liposomes and this system is capable of coupling succinate oxidation with oxygen reduction to water. This "strict anaerobic" bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and / ., anaerobes are much more complex than originally thought, o, .-~ 1. Cypionka IL Annu.Rev.Microbiol. 54, 827-480 (2000) 2. Lemos RS et al, FEBSletters, 24828, 1-4 (2001) 3. Lemos RS et al, Journal of Bioenergetics and Biomembranes., in press This work was supported by FCT-Portugal grants 36560/99 and 36558/99 to MT. R.S.Lemos is recipient of PRAXIS XXI grant BD / 19867 / 99
A strongly coupled diiron / radical state in ribonucleotide reductase mutant Y122H of E. coil studied by 57Fe- and tH / 2H- ENDOR F. L e n d z i a n a, M. K o l b e r g a, G. Bleifuss a, G. L a s s m a n n a, A. Grfislund b, and W. L u b i t z a ~Max- Volmer-Laboratorium fur Biophysikalische Chemie, Technische Universitat Berlin, PC14, Strasse des 17. Juni 135, 10623 Berlin (e-mail: lendzian@echo, chem.tu-berlin.de) bDepartment of Biophysics, Stockholm University, Arrhenius Laboratories. S-10691 Stockholm, Sweden Ribonucleotide reductase (RNR) catalyses the reduction of ribonucleotides, which is crucial for DNA synthesis. Subunit R2 of RNR carries in the acitve state an anti-ferromagnetically coupled ~t-oxo bridged di-ferric center (FemFem) and a stable, weakly coupled tyrosyl radical, Y122", which are both essential for the catalytic activity. The tyrosyl radical is generated from the FeHFen state in a reaction with molecular oxygen, which is similar to the oxygen activation reaction in other diiron oxygen enzymes, e. g. methane monooxygenase. Here we report on a new paramagnetic center observed in R2 mutant Y 1221-I, where tyrosine 122 is replaced by a histidine, inducing changes in the diiron ligand sphere. The high-field EPR and 57Fe-ENDOR results indicate that this center is a S=1/2 state consisting of FetUFem and a strongly coupled, probably ligated, radical ~. The radical is identified as phenylalanine by experiments on selectively phenylalanine-deuterated samples. 1. M. Kolberg, G. Bleifuss, S. P6tsch, A. Gr~islund, W. Lubitz, G. Lassmann, and F. Lendzian J. Am. Chem. Soc., 122, 9856 (2000) Work supported by Deutsche Forschungsgemeinschaft (La 751/3-1, Le 812/1-2)
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Journal of lnorganic Biochemistry 86 (2001)
315
Towards functional mimics of manganese superoxide dismutases Elizabeth A. Lewis a, John R. Lindsay Smith a, Paul H. Waltona, Hicham Khodr b, Robert C. Hiderb, Stephen J. Archibalda and Gerard M. Giblinc <'Department of Chemistry, University of York, Heslington, York YOlO 5DD, UK (email. [email protected]) hDepartment of Pharmacy, Franklin Wilkins Building, King's College London, 150 Stamford Street, London, SE1 8A W CMedicinal Chemistry, Glaxo Smith Kline Research and Development, Gunnels Wood Road, Stevenage, Herts., SGI 2NY An overproduction of superoxide has been implicated as the cause of many inflammatory processes, reperfusion injury and degenerative diseases. As a result, there is a great deal of current interest in the design of functional models of superoxide dismutases (SODs), enzymes which play an important role in protecting cells through catalysis of superoxide disproportlonatlon. R~N--U'2----7"-. N/~R This work involves the condensation and subsequent reduction of H /~"'/ H substituted salicylaldehydes with cis,cis-l,3,5-triaminocyclohexane (tach) to NH 1 HI_ ~ form a series of tri-amine ligands (1) and their corresponding Mn(III) complexes. Systematic variation of the substituent, X, provides a mechanism R R= for tuning both the solubility and electronic properties of the complexes. 2 The relationship between the X substituent, metal-based redox behaviour, Mn(III) X=H,CH30,CH3,CI,NO2,N(CH3)3 +,SO3" X binding and SOD activities of these systems has been investigated with the ultimate goal of designing an effective SOD mimic. !. 2.
Riley D., Chem Rev., 99, 2573-2588, (1999) Lewis E. A., Lindsay Smith J. R., Walton P. H., Archibald S. J., Foxon S. and Giblin G. M., J. Chem. Soc., Dalton Trans., 1159-1161 (2001)
Six-coordinate and five-coordinate Fen(CN)2(CO)x-thiolate complexes (x = 1, 2): synthetic advances for iron site of [NiFe] hydrogenases Wen-Feng Liaw, Jiun-Hung Lee, Hung-Bin Gau, Chien-Hong Chen, Shiou-Ju Jung, Wen-Yuan Chen, Ching-Han Hu, Chen-Hsiung Hung. Department of Chemistry, National Changhua University of Education, Changhua, 50058, Taiwan ([email protected]) The dicyano-dicarbonyl iron(II)-thiolate complexes trans, cis-[(CN),(CO)2Fe(S, S-C-R)]- (R = OEt (2), N(Et)2 (3)) were prepared from reaction of [Na][S-C(S)-R] and [Fe(CN)2(CO)3Br]- (1) obtained from oxidative addition of cyanogens bromide to [Fe(CO)4(CN)] respectively. Photolysis of THF solution of complexes 2 and 3 under CO atmosphere led to formation of the coordinate-unsaturated iron(II)-dicyanocarbonyl thiolate compounds [(CN)2(CO)2Fe(S , S-C-R)] (R = OEt (4), N(Et)2 (5)) respectively, the potential iron-site structural and functional model compound of active site of [NiFe] hydrogenases. The IR (CN) stretching frequencies and patterns of complexes 4 and 5 unambiguously identified two CN- ligands occupying cis positions. The theoretical calculations suggest that the architecture of five-coordinate complexes 4 and 5 with a vacant site trans to the CO ligand and two C N ligands occupying cis positions serves as a conformational preference. Complexes 2 and 3 were reobtained when THF solutxon of complexes 4 and 5 were exposed to CO atmosphere at ambient temperature individually. Isotopic shift experiments demonstrate the lability of carbonyl ligands of complexes 2, 3, 4 and 5. The facile formation of five-coordinate complexes 4 and 5 implicate that the strong mdonor, weak n-acceptor CN- ligands play a key role in creating/stabilizing five-coordinate iron(lI) complexes 4 and 5 with a vacant site trans to the CO ligand.
316
Journal of lnorganic Biochemistry 86 (2001)
Characterization of the particulate form of methane monooxygenase ( p M M O ) from methylococcus capsulatus (Bath) and methylosinus albus BG8 Raquel L. Lieberman, Peter E. Doan, Brian M. Hoffman, Amy C. Rosenzweig Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, IL 60208, U.S.A. ([email protected]) The conversion of methane to methanol is achieved in Nature by methane monooxygenase (MMO), an enzyme found in methanotrophic bacteria. This transformation is amazing because the enzyme selectively oxidizes the -100 kcal/mol C-H bond in methane at ambient temperature and pressure. All methanotrophs utilize the particulate form (pMMO), whereas some can also express the well-characterized soluble form (sMMO). It is currently believed that the active site o f p M M O contains copper and possibly iron, but the details of the nuclearity of the metal cluster, the identity and arrangement of the ligands, and the electronic environment have not been well established. We have purified pMMO from M. capsulatus (Bath) to homogeneity. This methanotroph expresses both the soluble and particulate form of methane mofiooxygenase. We are also purifying pMMO from M. albus BG8, a methanotroph that expresses only the particulate form. The pMMO enzyme from M. capsulatus (Bath) contains three subunits of molecular masses 29, 45, and 31 kDa in a stoichiometry of c~213:'{2. Reverse-phase HPLC data show that the [3 subunit is the least hydrophobic, followed by y and c~, respectively. We measure up to six copper ions per 200 kDa enzyme in our purified samples. All of the copper ions are EPR silent, suggesting that they are present as Cu(I).
Structural studies on desulfovibrio desulfuricans A T C C 27774 multiheme nitrite reductase - characterization of the subunits L. M. Lima Gongalves a,b, C. Cunha a, G. Almeida a, S. Macieira a, C. Costa a, j. Lampreia a, M. J. Romeo a, j. j. G. Moura a, I. Moura a a Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de CiFncias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte de Caparica, Portuga.l oInstituto Superior de Cidncias da Sa~de-Sul, Campus Universitdrio - Quinta da Granja, 2825-511 Caparica, Portugal (e-mail: [email protected]) Multiheme cytochrome c nitrite reductase (NiR) isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 is a membrane bound enzyme that catalyses the dissimilatory nitrite reduction to ammonia in a six-electron step. Initially the enzyme was described as a 66kDa monomeric protein, containing six e type hemes as prosthetic groups. 1 Later on, it was shown that NiR is a hetero-oligomeric complex composed by two subunits (63kDa and 19kDa) both containing c-type hemes z- the large subunit contains 5 heroes and the small most probably 4 hemes. The primary sequence of the two subunits are presented, discussed and compared with the D. vulgaris genomic data. We also report biochemical and spectroscopic studies on the two isolated subunits. So far, two 3D structures of the 5-heine containing large subunit o f NiR are available.4'5 The structure ofD. desulfuricans NiR was recently solved and.refined at 2.30 ~,. 3 1- Liu, M.-C., Costa, C. and Moura, I. Meth. Enz. 243,303-319 (1994). 2- Almeida, G. etal. J. Inorg. Biochem. 74, 63 (1999). 3- Dias, J.M. et al. Acta Crystallographica D56, 215-217 (2000). (preliminary results) 4- Einsle, O. et al. Nature 400, 476-480 (1999). 5- Einsle, O. et a.l J.Biol. Chem. 275, 39608-39616 (2000). We acknowledge the Fundaq~o para a Ci~ncia e a Tecnologia for the financial support.
Journal of Inorganic Biochemistry 86 (2001)
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Model complexes to mimic the 7t-stacking interaction in galactose oxidase X i a o m i n g L i u a n d M a l c o l m A. H a l c r o w *
School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK, (e-mail." x. [email protected], uk) Galactose oxidase (GOase) is a type II copper enzyme from wood-rot fungi that catalyses the oxidation of primary alcohols to aldehydes by molecular oxygen] An oxidisable tyrosine residue in GOase enables this enzyme to catalyse a two-electron oxidation reaction. The single crystal X-ray structure shows a square-pyramidal centre with two phenoxide donors (Figure 1). 2 The basal tyrosinate ligand is chemically modified with an ortho-thioether linkage formed by an oxidative cross-linking Tyr495 reaction to a cysteine side-chain, and is involved in a n-stacking interaction with a neighbouring indole ring. To further understand how the structural features of the GOase radical contribute to its chemistry, 3 we have synthesised new phenols based on the dibenzo[c,h]bicyclo-[4.4.1]undeca-3,8-dien-ll-one skeleton. 4 Acetalation of the ketone group in these compounds causes a conformational change, from an open boat-chair to a closed form in which the two annelated His496 Acetate Cys228 rings overlie each other. Comparison of the spectroscopic, structural and redox chemistries of the four derivatives in Figure 2 and their complexes has allowed Figure 1. Crystal structure of the us to understand in some detail how a thioether substituent and rt-rt interaction each contribute to the properties of a phenoxyl radical. GOase active site. 2
1.
Kaim W. and Rall J., Angew Chem., Int. Ed.
O
n1343601996 2. Ito N., et al, Nature, 350, 87-90 (1991). 3~ Halcrow M.A., et al, J. Chem. Soc., Dalton Trans., 4025-4036 (1997). 4, Mataka S., et al, Synlett., 1211-1227 (2000). We thank EPSRC (XL) and the Royal Society (London, MAH) for financial support.
Antitumor
HO~ O H
OH R = H, SMe OH F i g u r e 2. N e w p h e n o l l i g a n d s p r e p a r e d in this
platinum complexes bend adjacent purines with monofunctional binding
trans
Y a n g z h o n g L i u a, M a r i a F. S i v o b, G i o v a n n i N a t i l e b, a n d E i n a r S l e t t e n a
"Department of Chemistry, University of Bergen, Allegt. 41, N-5007 Bergen, Norway (e-mail. einar.sletten@kj, uib.no) bDepartment of Pharmaceutical Chemistry, University of Bari, Via E. Orabona 4, 1-70125,.Bari Italy. The new antitumor drug trans-EE binds to DNA forming essentially monofunctional adducts which appear to be responsible for the anticancer activity. We have previously shown that a DNA oligomer can be bent 45 degree towards the minor groove by trans-EE mono-binding 1, such a binding is comparable, in size, to that given by cisplatin which chelates on adjacent purines and bends DNA towards the major groove. A question that has arisen from this investigation, is how this mono-binding could bend DNA to such an extent? f Three monofunctional adducts of trans-EE with the nucleotide dimers, rApG, dApG and -~"~ . dGpA were synthesized. The kinetic processes were monitored by HPLC and adduct structures were studied by NMR spectroscopy. Guanine N7 was the only binding site in each adduct and in all of them a large down field shift of the G H8 signal was observed. The models were built using Insight II and were subsequently subject to energy minimization and molecular dynamics (BIOSYM) calculations using distance constraints extracted from the ROESY maps. The structure shows that the adenine base is bent 34 degree away from its initial geometry in the rApG adduct. The bending is probably caused by the hydrogen bond between a trans-EE imino rApGadduct proton and the adenine N7. 3.1 A between two nitrogen is suitable to form this hydrogen binding. The conversion to bifunctional adducts was also studied. 1. Andersen B., Margiotta, N., Coluccia M. Natile G. and Sletten E. Metal-Based Drags 7, 23-32 (2000)
318
Journal of Inorganic Biochemistry 86 (2001)
First examples of luminescent cyclometallated iridium(III) complexes as labelling reagents for biological substrates Kenneth Kam-Wing Lo,* Dominic Chun-Ming Ng and Chi-Keung Chung Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, KowIoon, Hong Kong, P.R. China We report the syntheses, photophysical and electrochemical properties of two novel cyclometallated iridium(III) complexes [Ir(ppy)2(phen-NCS)](PF6) (1) and [Ir(ppy)z(phen-NHCOCHzI)](PF6) (2) (Hppy = 2-phenylpyridine, phenNCS = 5-isothiocyanato- 1,10-phenanthroline, phen-NHCOCHzI - 5-iodoacetamido- 1,10-phenanthroline). These two iridium(III) complexes exhibit intense and long-lived orange-yellow luminescence upon photoexcitation in CH3CN at 298 K (1: ~-em = 608 nm, "Co = 0.41 p.s; 2: ~-em : 590 rim, Zo = 0.59 Its). The complexes show reversible metal-based oxidation couples at ca. +1.26 V vs. S.C.E. in CH3CN at 298 K. The first irreversible reduction waves of 1 and 2 occur at ca. - i . 2 1 and -1.08 V, respectively. The emission of the complexes in room-temperature fluid solutions is assigned to originate from a metal-to-ligand charge-transfer [dT~(Ir) --~ 7z*(diimme)] triplet excited state. Since the isothiocyanate and iodoacetamide groups can react with the primary amine and sulfhydryl groups, respectively, 1 and 2 have been used to label a universal M13 reverse sequencing primer modified with an amine or a sulfhydryl group at the 5'-end. The photophysical and hybridisation properties of the labelled oligonucleotides have been investigated. In addition, these two complexes have also been coupled to human serum albumin. The bioconjugates 1-HSA and 2-HSA display intense and long-lived emission at Xcm = 562 nm ['tl - 0.90 ~ts (33 %), "~2= 0.15 Its (67 %)] and ~em : 570 nm [rl = 0.66 ~ts (19 %), z2 0.07 Its (81%)], respectively, in degassed 50 mM Tris-C1 pH 7.4 at 298 K. In the presence of oxygen, the emission lifetimes exhibit only a small decrease, suggesting that the labels are shielded in the interior of the protein matrix. We acknowledge financial support from the Hong Kong Research Grants Council and the City University ofHong Kong.
An artificial hemoprotein: m i m o c h r o m e IV Angela Lombardia, Flavia Nastria, Omella Maglio a, Daniela Marascoa, Massimiliano Colettab, Vincenzo Pavonea Department of Chemistry, University of Naples "Federico II", via Cynthia 45, 1-80126 Naples, [TALY (e-mail: [email protected]) b Department Experimental Medicine and Biochemical Sciences, University ofRoma Tor Vergata, Via di Tor Vergata 135, 00133 Roma Peptide-based metalloprotein models have recently emerged as an efficient tool for reproducing the active site properties and reactivity. We have applied this approach to the development of a new class of ~ A ' ~ hemoprotein mimetics, named mimochromes ~. They are C2 simmetric dimers made up by a deuteroporphyrin ring covalently linked to two helical peptide chains; the heme iron is axially ligated by one or two His residues. The prototype, mimochrome I, contains two nonapeptides, which represent the smallest sequence able to fold into a-helical conformation and to cover the heine plane. The structural characterization of mimochrome I iron and cobalt complexes confirmed the design, even though it revealed some unexpected features. The peptide chains were able to fold up around the porphyrin in two different fashions, giving rise to A and A diastereomers. In order to drive the folding of mimochromes into a unique diastereomer, we further refined the structure by design. One approach was the substitution of amino acids in key positions, which may provide intra-molecular inter-helical interactions and stabilize the sandwich. This approach led to mimochrome IV, which was fully characterized in both the apo- and metal-forms. All the experimental results indicate a unique topology ofpeptide chains, as designed. 1.
Nastri F., Lombardi A., D'Andrea L., Sanseverino M., Maglio O. and Pavone V., Biopolymers, 47, 5-22 (1998)
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Journal of Inorganic Biochemistry 86 (2001)
319
Miniaturized metallo proteins: coordination properties of METP2 A n g e l a L o m b a r d i a, F l a v i a N a s t r i a, D a n i e l a M a r a s c o a, O m e l l a M a g l i o a, E n z o L a u r e n t i b, R o s a Pia Ferrari b, V i n c e n z o P a v o n e a
"Department of Chemistry, University of Naples "Federico II', via Cynthia 45. 1-80126 Naples, /TAL Y (e-mail. iombardi@chemistry, unina.it) b D e p a r t m e n t o f C h e m i s t r y I . F . M . , U n i v e r s i t y o f T o r i n o , 1-10125 T o r i n o , I t a l y The search for model systems able to reproduce the properties of natural metalloenzymes is one of the most pursued fields in bioinorganic chemistry. Synthetic metal complexes can be useful to provide information on the mechanism of action of natural systems. By changing the nature of the ligand, is possible to explore and rationalize the factors that tune the properties of the metal, in a way that could be easier respect to the use of mutant proteins. With the aim of obtaining peptide-based metalloprotein mimetics, we have recently employed a miniaturization process in the design of peptides, able to bind metal ions in a tetrahedral geometry. We developed a suitable prototype, METP (Miniaturized Electron Transfer Protein), which was designed using the mbredoxin as template structure. METP contains a Cys4 metal binding site and is made up of two identical undecapeptides self assembled upon metal ion coordination. It was shown to bind, in a tetrahedral geometry, Co(II), Fe(II)/Fe(III), Zn(II), as expected 2. With the aim of construct different tetra-coordinated metal-binding sites, we synthesized new molecules, which differ from the prototype METP for the nature of the coordinating residues. METP2 encloses a N4 metal binding site; its coordination properties, as ascertained by several spectroscopic techniques, will be presented. i. 2.
DeGrado W.F., Summa C.M., Pavone V., Nastri F., Lombardi A., Annu. Rev. Biochem. 68, 779-819(1999). Lombardi A., Marasco D., Maglio,O., Di Costanzo L., Nastri F., Pavone V. Proc. Nat. Acad. Sci. USA 97, 1192211927 (2000)
The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
The effect of polyamines (PA) on the coordination mode in Cu(II) complexes with adenosine 5'-triphosphate (ATP) and cytidine 5'-triphosphate (CTP) Lechoslaw Lomozik,Anna Gasowska Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland (e-mail. [email protected]) A study of the mode of Cu(II) coordianation with polyammes was performed for the systems Cu(II)/ATP(or CTP)/PA; (PA = putrescine (Put), 1,3-diammopropane (tn), spermidine (Spd), 3,7-diamino-4-azaheptane (3,3-tri), spermine (Spm) and 1,11-diamino-4,8-diazaundekane (3,3,3-tet)). The results proved the formation of heteroligand molecular compounds, protonated complexes and CULL' type complexes, each with a different type of metallation being a result of the interaction with polyamines. In the parent complexes Cu(ATP) and Cu(CTP), the copper(II) ions are coordinated through the oxygen atoms of the phosphate groups and the nitrogen atoms of the purine or pyrimidine rings ({N,O} chromophore). In the mixed complexes, the interactions of the polyamine with the anchoring complexes Cu(ATP) o r Cu(CTP) lead to an increase in the coordination efficiency of the phosphate groups of the nucleotides. In molecular complexes, e.g. Cu(ATP)H3(Spd) and Cu(CTP)H2(Put), the fully protonated polyamine is involved in noncovalent interactions with the donor centres of the purine or pyrimidine rings and it is located outside the inner coordination sphere. The metallation occurs through oxygen atoms of the phosphate groups (and in the case of ATP through the donor atom N(7) as well). In the protonated species, e.g. Cu(ATP)H2(Spd) and Cu(CTP)H3(Spm), the copper ions are coordinated by the oxygen atoms of the phosphate groups and deprotonated nitrogen atoms of the polyamine ({N×,O} chromophore). The complex is additionally stabilised as a result of intramolecular non-covalent interactions of the protonated amine groups of the polyamine with the donor centres of the nucleotide. It has been found that in the mixed-ligand CULL' type complexes, e.g. Cu(ATP)(3,3-tri) and Cu(CTP)(3,3,3-tet), the inner coordination sphere of the central atom comprises the oxygen atoms of the phosphate group from A T P o r CTP and the nitrogen atoms of the polyamine, whereas the donor nitrogen atoms from the nucleotide remain in the outer coordination sphere. Results of the studies of the metallation and non-covalent interactions in the model systems point to significant differences in the properties of the polyamines studied, which explains a higher biological activity of putrescine, spermidine and spermine compared to their homologues. The Polish State Committee for Scientific Research (KBN) is acknowledged for financial support.
Journal of lnorganic Biochemistry 86 (2001)
320
Energy tranduction performed by a monomeric tetrahaem cytochrome Ricardo O. Louro a. Teresa Catarino a'b, Jean LeGall c, David L. Turner a'd, Ant6nio, V. Xavier a a lnstituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6, Apt. 127. 2780 Oeiras, Portugal. Fax. +351-21-4468766.(e-mail: [email protected] b Departamento de Quimica da Faculdade de Ciencias e Tecnologia da Universidade Nova de Lisboa, Monte da Caparica, Portugal c Department of Biochemistry and Molecular Biology, Universityof Georgia, Athens GA 30602. USA d Department of Chemistry, University of Southampton, Southampton S017 1Bd, UK. In order to understand the mechanisms of biological energy transduction, it is essential to determine the microscopic thermodynamic parameters which describe the properties of each of the various centres involved in the reactions, as well as their interactions. These interactions between centres can then be interpreted in the light of structural features of the proteins. Redox titrations of cytochrome c3 from Desulfovibrio desulfuricans ATCC27774 followed by NMR and visible spectroscopy were analysed using an equilibrium thermodynamic model. This characterisation allows the identification of several pairs of centres for which there are clear conformational (non-Coulombic) contributions to their coupling energies, thus establishing the existence of localised redox- and acid-base-linked structural modifications in the protein (mechano-chemical coupling) ~. The modulation of interactions between centres observed for this cytochrome favours a two-electron transition coupled with the acid-base transition which is important for the physiological function of this protein. These physiologically relevant coupling patterns are not easy to engineer by mutagenesis even when using structurally homologous proteins 2. I. Louro, R.O., Catarino, T., LeGall, J., Turner D.L.,and Xavier, A.V. submitted 2. Salgueiro, C.A., da Costa, P.N., Turner, D.L. Messias, A.C., van Dongen, W.M.A.M., Saraiva, L.M. and Xavier, A.V. Biochemistry in press (2001) The Ministero della Ricerca Scientifica e Tecnologica (MURST) of Italy is acknowledged for financial support.
Designing metalloenzymes containing heteronuclear metal assembly Yi Lu,.Jeffrey A.
Sigman, Alan Gengenbach, Xiaotang Wang, Thomas D. Pfister, Hyeon K. Kim
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA (e-mail: [email protected]) Recent studies have uncovered increasing number of metalloenzymes containing heteronuclear metal assembly, such as the heme-manganese center in manganese peroxidase (MnP) and heine-copper center in heme-copper oxidase (HCO). MnP is a heme peroxidase that is involved in biodegradation of lignin and bioremediation of aromatic pollutants, such as polychlorinated biphenyls. HCOs are a superfamily of terminal oxidases in the respiratory chains. Compared with homonuclear metal centers, these heteronuclear metalloenzymes are less well-understood due to their inherent complexity that results from the multiple metal ions. They also pose significant challenges for protein design. We have been able to use stable, easy-to~¢¢ ','" :~i \ produce, and well-characterized proteins (such as cytochrome c peroxidase -~ ~ V : r C;- -" (CcP) and myoglobin (Mb)) as scaffolds for making biomimetic models of MnP ~ and HCOf Using these simple protein models, we are learning the principle of how these metal-binding sites are constructed to perform their functions and how they can be t-me tuned to realize different structural and functional properties. Latest results from these projects will be presented and their implication related to the structure and function of MnP and HCO will be discussed.
HCO (thin) and CusMb (thick) 1.
B.K.-S. Yeung et al. Chem. & Biol. 4, 215 (1997); b) A. Gengenbach et al. Biochemistry, 38, 11425 (1999). 2. J.A. Sigman et al. J. Am. Chem. Soc. 121, 8949 (1999); b) J. A. Sigman et al. J. Am. Chem. Soc., 122, 8192 (2000). We thank the National Institute of Health of the United States of America for Financial support.