- Email: [email protected]
Abstracts Poster Presentations (H)
Journal of lnorganic Biochemistry 86 (2001)
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X-ray resonance raman spectroscopy of metals in enzymes Stephen P. Cramera'b, Uwe Bergmanna'b, Stephan Friedrichb, Tobias Funk b, Pieter Glatzel~, Weiwei Gu~,Ping-chang Liua, Daulat Patilb, Hongxin Wanga a Department o f A p p l i e d S c i e n c e , UCDavis, CA 95656 (email: [email protected]) b Lawrence Berkeley National Lab, Berkeley, CA 94022 X-ray resonance Raman spectroscopy (XRRS) is a powerful new tool for studying the molecular, electronic, and magnetic structure of metalloenzyme active sites. Just as in the uv-visible technique, the XRRS experiment involves two monochromators that scan the intensity as a function of both scattered and excitation energy (Figure). Although metals in proteins are dilute, with current synchrotron sources there is sufficient photon flux to 'see' the metal centers. We have examined the Ni-Fe sites in hydrogenase [1], CO dehydrogenase [2] and the oxygen-evolving Mn complex of photosystem II [3]. We will present XRRS as well as L-edge, and x-ray MCD data addressing the metal electronic structure under different conditions. Metalloproteins are frequently radiation and vacuum sensitive, and maintaining sample integrity is critical for bioinorganic x-ray spectroscopy. Our attempts to improve detection efficiency with larger solid angle crystal analyzers, higher resolution superconducting detectors, lower sample temperatures, and higher magnetic fields will be presented. In this vein, we will try to quantitatively compare the relative merits of soft (L-edge) and hard x-ray (RIXS) experiments for elucidation of electronic structure. 1. 2. 3.
Wang, H.; ..., Cramer, S. P. J. Am. Chem. Soc.,, 122, 10544-10552 (2000) Ralston, C.; ..., Cramer, S. P. J. Am. Chem. Soc.,, 122, 10553-10560 (2000). Bergmann, ..., Cramer, S. P. J. Phys. Chem. B, 102, 8350-8352, (1998)
The National Institutes of Health (GM44380) and the Department of Energy (OBER) are acknowledged for support.
Characterization of the metal-binding site from ImiS, the metallo-~3-1actamase from Aeromonas veronii bv. sobria Patrick A. Crawford, Daniel N. Sobieski, Kelly A. Ashton, Michael W. Crowder Department of Chemistry and Biochemistry, Miami University, 112 Hughes Hal, 45056, Oxford, USA Aeromonas veronii bv. sobria is a water-borne, gram-negative bacterium which has been implicated in human wound and enteric infections and can be found in numerous water sources including treated drinking water. Treatment of A. sobria infections is complicated due to the presence of three [~-lactamases, AmpS and CepS both of which are serine-Clactamases and the metallo-[3-1actamase ImiS. ImiS is a Bush group 3b metallo-[3-1actamase, which requires divalent metal, usually zinc, for optimal activity. Like other group 3b metallo-[3-1actamases, ImiS is a carbapenemase, preferentially hydrolyzing carbapenems, [3-1actams that are not efficiently hydrolyzed by other metallo-13-1actamases. A significant structural difference exists between ImiS, and other group 3b metallo-13-1actamases, and the majority of metallo-[3-1actamases. Aside from the afore mentioned substrate profile difference, the most notable difference is that the majority of metallo-[3-1actamases require two divalent metal ions for optimal activity, while ImiS requires only one divalent metal ion for optimal activity, and the forced binding of a second equivalent of divalent metal is inhibitory. These differences are most likely due to fact that ImiS contains a single amino acid difference, His to Asn, from the consensus metallo-13-1actamase metal-binding ligands. In an effort to characterize the preferred metal-binding site of ImiS, Co(II)-substituted ImiS has been prepared and electronic absorption, EPR, and NMR spectra have been obtained. Also efforts have been undertaken to explore the changes, if any, to the preferred metal-binding site upon the addition of a second, inhibitory, divalent metal ion. In addition to characterizing the metal-binding site of ImiS, mechanistic studies have been undertaken to help elucidate the mechanistic differences between ImiS and other metallo-[3-1actamases in an effort to fred similar steps that could be targeted for the development of broad-spectrum transition-state analogue inhibitors.
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Reingeneering the metal coordination sphere in [3-1aetamase II from B a c i l l u s c e r e u s induces a p H - d e p e n d e n t conformational change Julia A. Cricco and Alejandro J. Vila Biophysics Section, University of Rosario, Suipacha 531, S2002LRK Rosario, ARGENTINA (e-mail: jcricco@fbioyf, unr. edu. ar) [3-1actamases are enzymes produced by bacteria which confer them resistance against [3-1actamantibiotics. Metallo-[3lactamases require one or two zinc(II) ions for their activity. They display a broad substrate profile, which includes penicillins, cephalosporins and carbapenems. 13-1actamase II from B. cereus, (13LII) can bind up to two zinc(II) ions, although only one of them (Zn 0 is required for its activity. Cys 168 residue is a ligand of Zn2 in I3LII, that is highly conserved in enzymes from other sources. Using site-directed mutagenesis techniques, Cys 168 residue was replaced by Glu in [3LII. C168E-[3LII displays an impaired catalytic activity compared to the WT enzyme at pH 6.0, that is partially recovered at higher pH values towards different substrates. The UV-Vis spectrum of Co(II)-C168E-13LII at high pH resembles that of Co(II)-13LII, whereas the low pH species exhibits an altered coordination geometry. This pH-induced change is not observed in WT Co(II)-[3LII. The ~HNMR spectra of Co(II)-C168E at pH 6 and 8 are markedly different. The IH NMR spectrum at pH 8 suggests the existence of a disordered active site. The alteration in the protonation state of the metal ion environment could promote a disordered active site similar to the one in the native enzyme. UNR, CONICET, ANPCyT and Fundaci6n Antorchas are acknowledged for fmancial support. JAC thanks CONICET for a graduate fellowship.
H y d r o p h o b i c interactions in a cyanobacterial plastocyanin - c y t o c h r o m e f c o m p l e x P._eterCrowley a, Gottfried Ottingb, Gerard W. Cantersa and Marcellus Ubbink a ~'Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands. bDepartment of Medical Biochemistry and Biophysics, Karolinska Institute, S-I 71 77, Stockholm, Sweden. (email: [email protected], nl) The complex of the photosynthetic redox partners plastocyanin and cytochromeffrom the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic ~ resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and to a lesser extent, the 'eastern face' of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f Rigid-body structure calculations using NMR derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex is independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH tends to reorganize the complex interface, while the binding affinity remains unaffected. Therefore protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.
Journal of lnorganic Biochemistry 86 (2001)
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Synthesis and characterization of some 3D metal ions complexes with Nsalicyliden-p-toluidine Liliana Cseh ~, I. Pantenburgb , C. Csunderlik ¢, Maria Mracec a, and Otilia Costisor a "Romanian Academy, Timisoara Branch, Inorganic Chemistry Laboratory, P. O. Box 5-612, R01900 Timisoara, Romania, e-mail. lili [email protected] blnstitute for Inorganic Chemistry, University of KOln, Greinstrasse 6, D-50939 KOln, Germany. CFaculty of Industrial Chemistry, University "Politehnica" Timisoara, 6, Bocsei str., RO-1900 Timisoara, Romania The synthesis of the mononuclear copper(II), cobalt(II) and iron(III) complexes with n-salicyliden-p-toluidine are presented. The nature of the complexes depends strongly on the metal to ligand ratio. Thus, the complexes of the type MeL2 were obtained and their structure proposed on the basis of elemental analyses, spectral properties and magnetic measurements. The structure of the copper(II) complex was established on the basis of single crystal X-ray spectroscopy. A theoretical study of the molecular and electronic structure of the ligand and its complexes were also performed using the molecular mechanics (MM+) and the PM3 methods from Hyper Chem package.
Comparison between functional models of 2-methyl-3-hydroxy-4(1H)oxoquinoline 2,3-dioxygenase (MeQDO) and quercetin 2,3-dioxygenase Mikl6s Czaun a, Gfibor Speier a'b ~Department of Organic Chemistry, University of VeszprOm, 8201 VeszprOm, HUNGARY (e-mail: [email protected]) bResearch Group for Petrochemistry, Hungarian Academy of Sciences, 8201 Veszpr~m, HUNGA R Y The base-catalyzed oxygenation of 2-phenyl-3-hydroxy-4(1H)-oxoquinoline (1) is a functional model of 2methyl-3-hydroxy-4(1H)-oxoquinoline 2,3-dioxygenase (MeQDO). This enzyme does not contain metal ions and is responsible for converting 2-methyl-3-hydroxy-4(1H)-oxoquinoline to the corresponding anthranilic acid derivatives. Products of two pathways were detected by . f~ gc-ms and the ratio of endoperoxide and dioxetane intermediates depends on the N solvents used (DMSO, DMF, THF, CH3CN ). Kinetic studies on the reaction resulted in the rate law -d[substrate] / dt = k [substrate] [02] [OH'] and EPR investigations ~ o [,--I evidenced the presence of an oxygen centered radical (g = 2.0059, a N = 1.69, a H = 1.07, a H, = 0.24, all,, = 0.24). We propose a mechanism, where the deprotonated substrate reacts with dioxygen in a SET reaction leading to superoxide ion and free ~ o organic radical. The flee radical reacts then with the superoxide ion (or dioxygen) in a rate-determining step. Compared this model reaction with those of quercetinase, which is a copper-containing metalloenzyme, we conclude that the difference in the stability of the isoelectronic radicals (1o is more stable than 2*) makes the involvement of CuII in
quercetinase necessary.
We thank the Hungarian Research Fund (OTKA T 030400) for support.
Journal of Inorganic Biochemistry 86 (2001)
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Studies on new tumour-active metal complexes Hassan Daghriri u, Philip Beale a, Fazlul Huq b Royal Prince Alfred Hospital, Gloucester House, Missenden Road, Camperdown, NSW 2050, Australia) b School of Biomedical Sciences C42, University of Sydney, East Street, Lidcombe, NSW 2141,Australia (e-mail: hdag6201@mail, usyd. edu.au) Although cisplatin is a widely used anticancer drug, it has a number of side-effects and also cancer cells develop resistance to the continued use of cisplatin. In attempt to reduce the side-effects and to widen the spectrum of activity, thousands of different platinum complexes have been prepared and tested. Since it is found that all cisplatin analogues in general have a similar spectrum of activity, it is logical to assume that in order to have a different spectrum of activity the designed complexes should have a structure different from that of cisplatin such that they may not obey the classical structure activity relationship. We have designed a number of heteronuclear metal complexes, some of which have shown activity against a number of ovarian and other cancer cell-lines. This poster describes the activities of the designed complexes and the nature of their interaction with DNA.
Interaction of lanthanide-DO3A complexes with adjacent glutamate carboxylate groups in Calbindin D9K Nicola D'Amelio bx, Silvio Aimea, Ivano Bertinib, Marco Fragaib, Yong-Min Lee b, Claudio Luchinat d and Gianni Valensin ° a Department b Department Italy CDepartment d Department
of Chemistry, I.F.M., University of Torino, via P. Giuria, 7, 1-10125, Torino, Italy of Chemistry, University of Florence, via L. Sacconi, 6, 50019, Sesto Fiorentino, of Chemistry, University of Siena, Via A. Moro, 53100, Siena, Italy of Soil Science and Plant Nutrition, University of Florence, Florence, Italy
The paramagnetic properties of lanthanides can be exploited for getting insight in specific parts of protein surface. Due to the high affinity of lanthanides for oxygen donors, carboxylate groups can be used as specific targets for the interaction. The DO3A ligand is particularly useful in these studies, for it coordinates lanthanides leaving two sites available for other donors. A solution of a 15N labeled sample protein, Calbindin D9K (75 residues) was titrated up to 200% with Gd3+-DO3A complex, and a combination of ~SN-ZHHSQC with inversion recovery experiment was used for measuring paramagnetic contributions to longitudinal relaxation rates after each addition. Relaxation data were used as distance constraints with different assumptions on the number of interacting complexes and their occupancies and the presence of four preferential interaction sites on the protein surface was inferred. Here we demonstrate that Gd(III)DO3A complex does interact with exposed carboxylates of Glu and Asp side chains. The interaction is apparently selective for pairs of close carboxylates gathered in one region thus providing an easy and quick tool for obtaining insight into the structural features of the protein.
Journal of Inorganic Biochemistry 86 (2001)
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Fur, an iron metalloregulatory protein from bacteria; reactivity with nitric oxide c
Beno~t d ' A u t r 6 a u x a, O l i v i e r H o m e r a, C l a u d i n e J e a n d e y a, J e a n - L o u i s O d d o u a, B e a t e B e r s c h , J e a n M a r c L a t o u r a, D a n i 6 l e T o u a t i b, I s a b e l l e M i c h a u d - S o r e t a. t/
S e r v i c e de Chimie I n o r g a n i q u e et Biologique, D R F M C / C E A - G r e n o b l e , 17 rue des Martyrs, 38054, G r e n o b l e cedex09, France. E m a i l ." d a u t r e a u @ d r f m c . c e n g . c e a . f r . b L a b o r a t o i r e de G~nOtique MolOculaire des R @ o n s e s Adaptatives, Institut J a c q u e s Monod, 2 p l a c e Jussieu, 75251, P a r i s cedex05, France. c L a b o r a t o i r e de R M N des Protdines, Institut de B i o l o g i e Structurale, 41 rue Jules Horowitz, 38042, Grenoble cedex09, France. The Fur protein is involved in the regulation of genes related to iron uptake in Gram-negative bacteria such as E. coli Fur is a dimer which has been proposed to bind iron as corepressor and then to act as a negative regulator via sequencespecific protein-DNA interactions at the promoter regions of iron regulated genes. The purified Fur protein is able to bind one metal dication (Co 2+, Mn 2+ and Fe 2÷) per monomer and also contains a tightly cysteines-bound zinc per monomer. Recently, Fur has been proposed, from in vivo experiments, to be inactivated by NO donors allowing then the expression of the flavohemoglobin gene which is under Fur control in Salmonella typhimurium 0). The interaction of NO and the Fur protein from E. coli has been investigated. In vivo and in vitro assays of the Fur activity in presence of NO together with the spectroscopic characterization of the iron site in absence (z) or in presence of nitric oxide will be present here. !~ 2,
Crawford, M.J.; Goldberg, D.E.; J. Biol Chem. 1998, 273, 51, 34028-34032 Jacquamet, L.; Dole, F.; Jeandey, C.; Oddou, J. L.; Perret, E.; Le Pape, L.; Aberdam, D.; Hazemann, J. L.; MichaudSoret, I.; Latour, J. M. J. Am. Chem. Soc. 2000, 122, 394-395.
Two blue copper proteins and their interaction with lipids and redox partners Eva Danielsena, Lars ElkjaerJorgensena " D e p a r t m e n t o f M a t h e m a t i c s and Physics, The R o y a l Veterinary and A g r i c u l t u r a l University, T h o r v a l d s e n s v e j 40, DK-1871 F r e d e r i k s b e r g C, D e n m a r k (e-mail. e l k j a e r @ k v l . d k and eda@kvl, dk) Cupredoxins can be encountered in several places in biology. It is a class of smaller (about 10-15 kD) globular [3barrel proteins containing a type I copper site. As part of their function in electron transfer, they form short-lived interaction complexes with their redox parmers. This is a study of the interaction of plastocyanin and amicyanin with their redox partners as well as with lipid membranes. The two cupredoxins have been picked since they function in similar environments (plastocyanin in the thylakoid lumen of plants and amicyanin in the periplasmic space of bacteria) allowing for a comparative study. The poster will contain binding studies between well-characterised lipid vesicles of DMPC and DGDG and proteins where the copper has been substituted with either radioactive cadmium or silver. Measurements include radioactive labeling, and Perturbed Angular Correlations (PAC)-spectroscopy for varying pH and redox states. The PAC experiments can measure the structure and binding of interaction complexes between metal-substituted copper proteins and their redox partner for varying pH, redox state, and ionic strength. Investigations were made on amicyanin+MADH, plastocyanin+PS 1 ~, and plastocyanin+cytochrome b6f. Some results include Observation of Ag+-plastocyanin binding to lipids at low pH Derivation of a dissociation constant of approximately 50 gM for the Cd2+-amicyanin+MADH complex Observation of two structural species of free amicyanin with different pH dependency 1.
Danielsen E. et al, Biochemistry, 38, 11531 - 11540 (1999)
The Research Training Network in the fifth programme of the European Commission, and the Danish Technical Research Council are acknowledged for financial support.
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Comparative Studies on Different Forms of Human Phenylalanine Hydroxylase
Sylvia Daoud Kinziea, Mui Sama, and Mahdi Abu-Omara ~Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Avenue, 90095, Los Angeles, California, U.S.A. (email: [email protected]) Human phenylalanine hydroxylase (hPAH) is a non-heme iron-dependent enzyme that catalyzes the hydroxylation of L-Phe into L-Tyr in the presence of tetrahydrobiopterin and molecular oxygen. Single base mutations or small deletions in the PAH gene result in phenylketonuria (PKU), an autosomal disorder of phenylalanine metabolism. In the present work, we have expressed and characterized recombinant hPAH in Escherichia coli using the pMAL and the thioredoxin expression systems. The wild-type form and three different deletion mutants (Thr117-Ser411, Thr117-Leu424, Thr117Lys452) were expressed with high specific activity. Each form revealed a different state of oligomerization as determined by gel filtration chromatography. The wild-type protein consists of active dimeric and tetrameric forms while the 117-411 is monomeric, the 117o424 dirneric and the 117-452 tetrameric. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) techniques to monitor folding and unfolding transitions reveal differences in the stability of the different forms. The melting temperature for the wild-type tetramer is 52 °C + 2 °C and that of the deletion mutants is 60 °C + 2 °C. However, no additional stabilization is seen in the presence of iron(II) which is absolutely required for catalytic activity. To further understand the structural stability and folding properties of the protein, we are examining the catalytic, folding and oligomerization defects of the PAH protein caused by several PKU and non-PKU mutations. The March of Dirnes Foundation is acknowledged for financial support.
Preferential formation of a 1,4-interstrand DNA crosslink rather than a 1,2intrastrand crosslink by a dinuclear platinum complex
Murray S. Daviesa, Susan J. Bemers-Pricea, John W. Coxb, Donald S. Thomasa, Nicholas Farrellb aDepartment of Chemistry, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, AUSTRALIA. (email: [email protected], edu.au) bDepartment of Chemistry, Virginia Commonwealth University, 1001 Main Street, Richmond, Virginia, 23284-2006, USA. Competition between formation of a 1,4-GG interstrand or a 1,2-GG intrastrand crosslink (in both 3',3" and 5',5' directions) has been examined by following the reaction between 15N-[{transPtCI(NH3)2} z(~t-NH2(CHz)6NH2)] 2+, ( 1,1/t,t (n=6)), and the duplex 5 'd(ATACATGGTACATA)-3".5"-d(TATGTACCATGTAT)-3" (I) by [~H,tSN] HSQC NMR spectroscopy. The reaction affords one major monofunctional adduct and two bifunctional adduct conformers. Analysis of the NOESY spectrum of the final product % confirmed the presence of two conformers of the 1,4-GG interstrand cross-link in the 5',5' direction. Monofunctional binding of the drug is considerably faster with (I) than with the 12 mer duplex, l 5"-{d(ATATGTACATAT)}2, but the rate of closure of the monofunctional adduct to form the bifunctional crosslink is similar. 1. Cox, J. W., Berners-Price, S. J., Davies, M. S., Qu, Y. and Farrell, N., J. Am. Chem. Soc., 123, 1316-1326 (2001). The authors thank the Australian Research Council, the U. S. National Institutes of Health, U. S. National Science Foundation and the American Cancer Society for t'mancial support.
Journal of Inorganic Biochemistry 86 (2001)
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Evidence of c o m p o u n d I and compound II ferryl species from the reaction of hemoglobin I from lucina pectinata with hydrogen peroxide Walleska De Jesfis-Bonilla b, Jos6 Cerda a, Juan L6pez-Garriga b, Eunice Ramirez-Mel6ndezb Department of Chemistry, Michigan State University, East Lansing, Michigan, USA b Department o f Chemisty, University of Puerto Rico, Mayagiiez Campus, P.O. Box 9019, 00681-9019, Mayagiiez, Puerto Rico (e-mail. wdejesus@sonw, uprm.edu) The formation of ferryl heme species has been identified as the main intermediates in heme protein peroxidative reactions.l'2 We report UV-Vis and stopped-flow kinetic measurements, which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce the ferryl species compound I and compound II. Compound I appears to be relatively stable displaying an absorption band at 648 nm. The rate constant value (k4t) for the oxidation of the ferrous HbI is 25 M l s 1, nearly four times smaller than the reported for myoglobin; the conversion of compound I to compound II (k23) is 3.0 x 10-2s1, more than a hundred times smaller than myoglobin; and the oxidation of the ferric heme (k12+k13) is 2.0 x 102 Mts -1. The k23/k4! ratio shows that HbI has a higher peroxidative capacity for removing H202 from solution in relation to the ferrous oxidation. In HbI from L. pectinata the stabilization of the ferryl species is due to the unusual collection of amino acid residues (Q64, F29, F43, F68) in the heme pocket active site of the protein. Resonance Raman studies confirm that the compound II ferry1 vibration frequency is present at 805 cm -1 for the HbIFeW=160, and at 769 cm-1 for the HbIFeIV=lSO species. This higher ferryl vibrational mode result implies the lack of hydrogen bonding between the Q64 and the ferryl species. This unusual vibrational mode also suggests that the carbonyl group of Q64 in HbI points toward the iron of the heme group, increasing the electron density in the heme pocket moiety. 1. 2.
Snyder S., Welty E., Walder R., William L. and Walder J., Proc. Natl. Acad. Sci. USA, 84, 7280-7284 (1987). Alayash A.I., Ryan B. A., Eich R.F., Olson J.S. and Cashon R.E., J. Biol. Chem., 274, 2029-2037 (1999).
This project was supported, in part, by NSF (MCB-9974961) and NIH (MBRS/SCORE-SO6GM08103-27).
Electron transfer between proteins: effect of cross-linking Thvra E. de Jongh,a Irene M.C. van Amsterdam,a Marcellus Ubbink,a Oliver Einsle,b Albrecht Messerschmidt, b Angelo Merli, c Davide Cavazzinif Gian Luigi Rossi ~ and Gerard W. Canters ~ a Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, the Netherlands b Max-Planck-Institutffir Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, 82152 Martinsried, Germany c Instituto di Scienze Biochimiche, Universita d e g l i s t u d i di P arm a, Parco della Scienze 23A, 143100 Parma, Italy In order to gain more insight into the relative orientation of proteins in short-lived complexes of redox proteins we have analysed the effect of cross-linking on the electron transfer between two molecules of the blue copper protein azurin, in the so-called electron self-exchange (e.s.e.) reaction. In the crystal packing of wild type azurin two molecules face each other with opposing hydrophobic patches, which is thought to be similar to the transient complex that is formed during the e.s.e, reaction in solution. The Asn42 residues of each monomer are in close proximity, which makes them, for the purpose of cross-linking, suitable for mutagenesis. Asn42 was replaced by cysteine and two different dimers were constructed: 1) a disulfide bridged dimer (Cys42-S-S-Cys42) and 0 0 2) a 'BMME-dimer', which is cross-linked using the bifunctional symmetric linker bismaleimidemethylether (BMME) creating a Cys42-S-BMME-S-Cys42 linkage. The The cross-linking electron transfer rates within, as well as between, the dimers were analysed by NMR. The results were complemented by the crystallographic data obtained of these constructs. reagent bis-
~ -CH~-O-CH~-N~
maleimidemethylether
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Journal of Inorganic Biochemistry 86 (2001)
Mutagenesis of the cytochrome c peroxidase from Rhodobacter capsulatus L i n a D e S m e t a, G r a h a m W. P e t t i g r e w b, Y v e s G u i s e z a a n d J o z e f V a n B e e u m e n a
a Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Ledeganckstr. 35, 9000, Gent, Belgium, b Department of Preclinical Veterinary studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall Edinburgh EH9 1QH, UK Cytochrome c peroxidases (CCP) have been found in yeast and bacteria. In bacteria two systems are available for the scavenging of potential harmful hydrogen peroxide: cytoplasmic catalase and periplasmic dihaem CCP. The latter catalyzes the oxidation of ferrocytochrome c and the reduction of H202. The reaction resembles that carried out by the haem b containing CCP from yeast, except that the BCCP's do not create a free radical during catalysis but, instead, use two haem c groups. These haems have totally different properties, one haem is low-potential and acts as the peroxidatic center, while the other is high potential and functions as the electron transfering haem. We were able to overproduce the Rb. capsulatus CCP in E. coli. The recombinant protein was purified in a four-step procedure. Diffraction-quality crystals were obtained under two conditions. The crystal structure of the dihaem cytochrome c peroxidase was determined by molecular replacement using Ps. aeruginosa as a model 1. The structure solved is that of the completely oxidized enzyme. In this form the N-terminal haem iron is ligated with histidines 55 and 74 and therefore the low potential haem, while the C-terminal haem c is ligated by histidine 205 and methionine 278. The completely oxidized enzyme is inactive. The reaction mechanism of the CCP's is rather complex and involves changes in iron ligands and spin states. The activity of the enzyme is also calcium dependent 2. Two possible models for hydrogen peroxide reduction by CCP have been proposed so far ~.3. In the first model a switch to a high spin state at the low potential N-terminal haem is the main event. In the second model both a spin switch and a ligand switch occurs. The N-terminal haem becomes the high-potential electron transferring haem, while the C-terminal haem becomes a penta-coordinate peroxidatic center. Mutagenesis was used to give more insight in the reaction cycle. We will present the structure and characterisation of the wild type enzyme and of several mutants. 1.
Fulop V.et al., Structure, 3, 1225-1233 (1995); 2. Gilmour R. et al., Biochem. J,. 300, 907-914, (1994); 3. Prazeres S. et al., J. Biol. Chem., 31, 24264-24269 (1995)
Does stereospecific oxidation by compound I of cytochrome P450 ever proceed in a concerted manner? Sam P. de Visser. Francois Ogliaro and Sason Shaik Department of Organic Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Density Functional B3LYP/LACVP calculations were performed on a Cytochrome P450 model Compound I, oxoiron porphyrin thiol [FeOPorSH]. The reaction path for the epoxidation of ethene by Compound I was studied, v3 It was found that the reaction proceeds stepwise via a radical intermediate, which can exist either in a low spin (doublet) or high spin (quartet) form. Additionally, the radical intermediate can have the iron in oxidation states Fem or Fe w. The formation of the radical complexes from reactants was found to proceed via barriers of 13.9 kcal mol 1 (4FelV) and 14.9 kcal mol l (2FelV). Both low spin radical complexes react in a barrierless fashion to give an epoxide complex. On the high spin surface small barriers of 2.3 kcal mol l (Fe TM) and 7.2 kcal mol l (Fe ul) leading to an epoxidation complex were obtained. In addition, we have calculated the barrier for synchronous attack of the ethene leading to the epoxide complex in a concerted manner. This barrier was found to be 21.1 kcal mo1-1. Consequently, the concerted reaction path is not available for this process. Therefore, the stereospecifity of P450 oxidation originates from the asynchronous mechanism in the doublet spin state whereas occasional stereochemistry and side products originate from the stepwise mechanism in the quartet spin state. I. 2. 3.
S.P. de Visser, F. Ogliaro, N. Harris and S. Shaik J. Am. Chem. Soc. 2001, 123, 3037. S.P. de Visser, F. Ogliaro and S. Shaik Angew. Chem. Int. Ed. 2001, in press. S.P. de Visser, F. Ogliaro and S. Shaik submittedfor publication.
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Biochemical characterization of the reductases involved in denitrification in the hyperthermophilic pyrobaculum aerophilum and purification of the NO reductase S. de V r i e s ", M . J . F . S t r a m p r a a d a, I. S c h r 6 d e r b
aKluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands bDepartment of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095-1489 The molecular biology and biochemistry of denitrification in Proteobacteria has been studied extensively (1). However, little is known about denitrification in Archaea. Pyrobaculum aerophilum is a hyperthermophilic Archaeon growing optimally at temperatures of 100°C utilizing nitrate or oxygen as terminal electron acceptors for heterotrophic growth. Previous work has indicated that nitrate is reduced via nitrite, NO and N20 to N2, i.e. in both Proteobacteria and Archaea the denitrification intermediates are the same. Our aim is to determine the structure and function of the four reductases NAR, NIR, NOR and NzOR involved in denitrification in P. aerophilum. To this end, we have developed both polarographic and colorimetric assays to determine the activities of the four reductases continuously up to temperatures of 95 °C enabling the rapid detection of active fractions obtained during purification. Enzyme assays with whole membranes using menahydroquinone (MQH2) as electron donor (the natural quinone in P. aerophilum is a (novel type of) menaquinone) with nitrate, nitrite, nitric oxide or nitrous oxide as acceptor showed that in P. aerophilum all four reductases are membrane bound, in contrast to Proteobacteria where NIR and N2OR are watersoluble enzymes residing in the periplasmic space together with small cytochromes and cupredoxins. P. aerophilum does not contain soluble c-cytochromes or cupredoxins. NOR was extracted with laurlymaltoside and after CM-sepharose- and HAP- chromatography purified to homogeneity. The purified NOR from P. aerophilum is single subunit enzyme displaying high MQH2: NO oxidoreductase activty upto 95 °C. The enzyme contains heme and non-heme iron in the ratio of 2:1. Using EPR and UV-Vis spectroscopy we showed that one of the hemes is a low-spin heme b like heme (gz= 3.015; gy= 2.226; g×= 1.45), the other is high-spin becoming low spin upon binding of CO.
Conformational transitions of polyAx2polyU in the presence of Mg2+ in solutions with different ionic strength. M.V. Dcgtyar, V.A. Sorokin, G.O. Gladchenko, V.A. Valeev, Yu.P. Blagoi B I. Verkin Institute for Low Temperature Physics and Engineering, National Academy of Sciences of Ukraine,Lenin ave. 47, 61164, Kharkov, Ukraine, (e-mail. my [email protected]) Heat induced conformational transitions of polyAx2polyU (Ax2U) in the presence of Mg 2÷ were studied by UV spectroscopy at solutions with different ionic strength: 10-~, 3x10 -2 and 10~M Na% At low ionic strength (10 .2 and 3x 10-ZM Na ÷) two transitions take place: a) Ax2U ~ AxU + polyU, b) AxU --, polyA + polyU. At 10-tM Na ÷ Ax2U dissociates directly to one stranded species. Full faze diagrams of conformational equilibrium in polynucleotides solutions are obtained. Increase of [Mg 2+] results in the increase of thermostability of all helix structures. Concentrational dependencies of 3--*2, 2--+1 and 3-~1 melting temperatures on [Mg 2+] permit us to calculate Mg 2+ binding constants using equilibrium binding theory [1]. At low binding degree the values of association constants (table 1) agree to the extent of 10% with data from [2] recalculated for .10.2 and 3 x 10.2 M Na + using slope
OlgK
_ -1.6.
0 ig[Na + ] Table 1. Binding constants of Mg 2+ interacting with Ax2U and AxU (K, M l). [Na +],M 10.2 3 x 10z 101 our data [2] our data [2] our data Ax2U 9x104 8x103 2x103 AxU
8x104
8.5×10 4
10 4
1.3x104
1 Lazurkin Yu.S., Frank-Kamenetskii M.D., Trifonov E.N. Biopolymers, 2, 1253 - 1306 (1970) 2. Ohyama T., Cowan I.A. JBJC, 1, 83-89 (1996)
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COME, the ontology for bioinorganic centres in proteins Kirill D e g t y a r e n k o , S e r g i o C o n t r i n o
EMBL Outstation, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CBIO 1SD, U.K. (e-mail. [email protected], uk) Annotation and interoperability of biological databases in the "post-genornic" era is seriously hampered by the lack of a common nomenclature for biochemical entities. The Gene Ontology Consortium I addresses the problem by developing ontologies for biological process, molecular function and cellular component. It is hoped that Gene Ontology (GO) will become the lingua franca for annotation of biological databases. However, the bioinformatics community has paid too little attention to the complex biochemical structures such as metalloproteins 2 and it also remains for the wealth of knowledge accumulated by bioinorganic chemistry to be organised in a systematic way. COME (Co-Ordination of Metals) represents the ontology for bioinorganic centres in complex proteins. COME consists of three types of entities: molecule (MOL), bioinorganic motif (BIM), and bioinorganic proteins (PRX), each entity is assigned a unique identifier. MOL entity represents "small molecule" (as opposed to macromolecule) which, in complex with a polypeptide, forms a functional protein. BIM consists of at least one centre (metal atom, inorganic cluster, organic molecule) and two or more endogenous and/or exogenous ligands. The PRX entity refers to the functional protein as well as separate protein domains and suhunits. It may include MOL, BIM as well as other PRX entities. The data are currently stored in XML format and are available from the web site 3. The relational implementation of the database is under development. 1. 2. 3.
The Gene Ontology Consortium, Nature Genetics 25, 25-29 (2000) Degtyarenko, K.N., Bioinformatics, 16, 851-864 (2000) http://www.ebi.ac.uk/-kirill/come/
New myoglobin reconstituted with an iron porphycene as a structural isomer of heme moiety Hirohisa Dejima
a,
Takashi Hayashi
a,b, T a k a s h i
M a t s u o b, Y o s h i o H i s a e d a a
~Department of Chemistry and Biochemistry, Kyushu University and bTOREST in Japan Science and Technology Corporation, Fukuoka 812-8581, JAPAN (e-mail: d e j i i t c m @ m b o x . n c . k y u s h u - u . a c . j p ) Myoglobin (Mb) is an oxygen storage protein containing protoporphyrin IX iron complex as a prosthetic group. To change the physiological and/or chemical property of the myoglobin, we prepared an iron mesoporphycene as a structural isomer of mesoporphyrin and then inserted it into apomyoglobin by usual method as shown in the following scheme. The reconstituted metMb exhibits a characteristic reconstitution absorption spectrum with peaks at 387 (Soret), 564, and 624 nm. ESI-TOF-MS spectrum of the reconstituted metMb shows two peaks at ngz = 17,577 and 16,959, corresponding to holoMb and apoMb, respectively. After the reduction of the metMb to deoxyMb, we monitered CO complex at 393 HOOC COOH (Soret) and 613 nm in pH 7.0, 100 mM phosphate buffer apomyoglobin iron porphycene involving CO gas. Here, we wish to report the characterization and property of the reconstituted Mb with the iron porphycene.
191
X-ray resonance raman spectroscopy of metals in enzymes Stephen P. Cramera'b, Uwe Bergmanna'b, Stephan Friedrichb, Tobias Funk b, Pieter Glatzel~, Weiwei Gu~,Ping-chang Liua, Daulat Patilb, Hongxin Wanga a Department o f A p p l i e d S c i e n c e , UCDavis, CA 95656 (email: [email protected]) b Lawrence Berkeley National Lab, Berkeley, CA 94022 X-ray resonance Raman spectroscopy (XRRS) is a powerful new tool for studying the molecular, electronic, and magnetic structure of metalloenzyme active sites. Just as in the uv-visible technique, the XRRS experiment involves two monochromators that scan the intensity as a function of both scattered and excitation energy (Figure). Although metals in proteins are dilute, with current synchrotron sources there is sufficient photon flux to 'see' the metal centers. We have examined the Ni-Fe sites in hydrogenase [1], CO dehydrogenase [2] and the oxygen-evolving Mn complex of photosystem II [3]. We will present XRRS as well as L-edge, and x-ray MCD data addressing the metal electronic structure under different conditions. Metalloproteins are frequently radiation and vacuum sensitive, and maintaining sample integrity is critical for bioinorganic x-ray spectroscopy. Our attempts to improve detection efficiency with larger solid angle crystal analyzers, higher resolution superconducting detectors, lower sample temperatures, and higher magnetic fields will be presented. In this vein, we will try to quantitatively compare the relative merits of soft (L-edge) and hard x-ray (RIXS) experiments for elucidation of electronic structure. 1. 2. 3.
Wang, H.; ..., Cramer, S. P. J. Am. Chem. Soc.,, 122, 10544-10552 (2000) Ralston, C.; ..., Cramer, S. P. J. Am. Chem. Soc.,, 122, 10553-10560 (2000). Bergmann, ..., Cramer, S. P. J. Phys. Chem. B, 102, 8350-8352, (1998)
The National Institutes of Health (GM44380) and the Department of Energy (OBER) are acknowledged for support.
Characterization of the metal-binding site from ImiS, the metallo-~3-1actamase from Aeromonas veronii bv. sobria Patrick A. Crawford, Daniel N. Sobieski, Kelly A. Ashton, Michael W. Crowder Department of Chemistry and Biochemistry, Miami University, 112 Hughes Hal, 45056, Oxford, USA Aeromonas veronii bv. sobria is a water-borne, gram-negative bacterium which has been implicated in human wound and enteric infections and can be found in numerous water sources including treated drinking water. Treatment of A. sobria infections is complicated due to the presence of three [~-lactamases, AmpS and CepS both of which are serine-Clactamases and the metallo-[3-1actamase ImiS. ImiS is a Bush group 3b metallo-[3-1actamase, which requires divalent metal, usually zinc, for optimal activity. Like other group 3b metallo-[3-1actamases, ImiS is a carbapenemase, preferentially hydrolyzing carbapenems, [3-1actams that are not efficiently hydrolyzed by other metallo-13-1actamases. A significant structural difference exists between ImiS, and other group 3b metallo-13-1actamases, and the majority of metallo-[3-1actamases. Aside from the afore mentioned substrate profile difference, the most notable difference is that the majority of metallo-[3-1actamases require two divalent metal ions for optimal activity, while ImiS requires only one divalent metal ion for optimal activity, and the forced binding of a second equivalent of divalent metal is inhibitory. These differences are most likely due to fact that ImiS contains a single amino acid difference, His to Asn, from the consensus metallo-13-1actamase metal-binding ligands. In an effort to characterize the preferred metal-binding site of ImiS, Co(II)-substituted ImiS has been prepared and electronic absorption, EPR, and NMR spectra have been obtained. Also efforts have been undertaken to explore the changes, if any, to the preferred metal-binding site upon the addition of a second, inhibitory, divalent metal ion. In addition to characterizing the metal-binding site of ImiS, mechanistic studies have been undertaken to help elucidate the mechanistic differences between ImiS and other metallo-[3-1actamases in an effort to fred similar steps that could be targeted for the development of broad-spectrum transition-state analogue inhibitors.
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Reingeneering the metal coordination sphere in [3-1aetamase II from B a c i l l u s c e r e u s induces a p H - d e p e n d e n t conformational change Julia A. Cricco and Alejandro J. Vila Biophysics Section, University of Rosario, Suipacha 531, S2002LRK Rosario, ARGENTINA (e-mail: jcricco@fbioyf, unr. edu. ar) [3-1actamases are enzymes produced by bacteria which confer them resistance against [3-1actamantibiotics. Metallo-[3lactamases require one or two zinc(II) ions for their activity. They display a broad substrate profile, which includes penicillins, cephalosporins and carbapenems. 13-1actamase II from B. cereus, (13LII) can bind up to two zinc(II) ions, although only one of them (Zn 0 is required for its activity. Cys 168 residue is a ligand of Zn2 in I3LII, that is highly conserved in enzymes from other sources. Using site-directed mutagenesis techniques, Cys 168 residue was replaced by Glu in [3LII. C168E-[3LII displays an impaired catalytic activity compared to the WT enzyme at pH 6.0, that is partially recovered at higher pH values towards different substrates. The UV-Vis spectrum of Co(II)-C168E-13LII at high pH resembles that of Co(II)-13LII, whereas the low pH species exhibits an altered coordination geometry. This pH-induced change is not observed in WT Co(II)-[3LII. The ~HNMR spectra of Co(II)-C168E at pH 6 and 8 are markedly different. The IH NMR spectrum at pH 8 suggests the existence of a disordered active site. The alteration in the protonation state of the metal ion environment could promote a disordered active site similar to the one in the native enzyme. UNR, CONICET, ANPCyT and Fundaci6n Antorchas are acknowledged for fmancial support. JAC thanks CONICET for a graduate fellowship.
H y d r o p h o b i c interactions in a cyanobacterial plastocyanin - c y t o c h r o m e f c o m p l e x P._eterCrowley a, Gottfried Ottingb, Gerard W. Cantersa and Marcellus Ubbink a ~'Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands. bDepartment of Medical Biochemistry and Biophysics, Karolinska Institute, S-I 71 77, Stockholm, Sweden. (email: [email protected], nl) The complex of the photosynthetic redox partners plastocyanin and cytochromeffrom the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic ~ resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and to a lesser extent, the 'eastern face' of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f Rigid-body structure calculations using NMR derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex is independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH tends to reorganize the complex interface, while the binding affinity remains unaffected. Therefore protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.
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Synthesis and characterization of some 3D metal ions complexes with Nsalicyliden-p-toluidine Liliana Cseh ~, I. Pantenburgb , C. Csunderlik ¢, Maria Mracec a, and Otilia Costisor a "Romanian Academy, Timisoara Branch, Inorganic Chemistry Laboratory, P. O. Box 5-612, R01900 Timisoara, Romania, e-mail. lili [email protected] blnstitute for Inorganic Chemistry, University of KOln, Greinstrasse 6, D-50939 KOln, Germany. CFaculty of Industrial Chemistry, University "Politehnica" Timisoara, 6, Bocsei str., RO-1900 Timisoara, Romania The synthesis of the mononuclear copper(II), cobalt(II) and iron(III) complexes with n-salicyliden-p-toluidine are presented. The nature of the complexes depends strongly on the metal to ligand ratio. Thus, the complexes of the type MeL2 were obtained and their structure proposed on the basis of elemental analyses, spectral properties and magnetic measurements. The structure of the copper(II) complex was established on the basis of single crystal X-ray spectroscopy. A theoretical study of the molecular and electronic structure of the ligand and its complexes were also performed using the molecular mechanics (MM+) and the PM3 methods from Hyper Chem package.
Comparison between functional models of 2-methyl-3-hydroxy-4(1H)oxoquinoline 2,3-dioxygenase (MeQDO) and quercetin 2,3-dioxygenase Mikl6s Czaun a, Gfibor Speier a'b ~Department of Organic Chemistry, University of VeszprOm, 8201 VeszprOm, HUNGARY (e-mail: [email protected]) bResearch Group for Petrochemistry, Hungarian Academy of Sciences, 8201 Veszpr~m, HUNGA R Y The base-catalyzed oxygenation of 2-phenyl-3-hydroxy-4(1H)-oxoquinoline (1) is a functional model of 2methyl-3-hydroxy-4(1H)-oxoquinoline 2,3-dioxygenase (MeQDO). This enzyme does not contain metal ions and is responsible for converting 2-methyl-3-hydroxy-4(1H)-oxoquinoline to the corresponding anthranilic acid derivatives. Products of two pathways were detected by . f~ gc-ms and the ratio of endoperoxide and dioxetane intermediates depends on the N solvents used (DMSO, DMF, THF, CH3CN ). Kinetic studies on the reaction resulted in the rate law -d[substrate] / dt = k [substrate] [02] [OH'] and EPR investigations ~ o [,--I evidenced the presence of an oxygen centered radical (g = 2.0059, a N = 1.69, a H = 1.07, a H, = 0.24, all,, = 0.24). We propose a mechanism, where the deprotonated substrate reacts with dioxygen in a SET reaction leading to superoxide ion and free ~ o organic radical. The flee radical reacts then with the superoxide ion (or dioxygen) in a rate-determining step. Compared this model reaction with those of quercetinase, which is a copper-containing metalloenzyme, we conclude that the difference in the stability of the isoelectronic radicals (1o is more stable than 2*) makes the involvement of CuII in
quercetinase necessary.
We thank the Hungarian Research Fund (OTKA T 030400) for support.
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Studies on new tumour-active metal complexes Hassan Daghriri u, Philip Beale a, Fazlul Huq b Royal Prince Alfred Hospital, Gloucester House, Missenden Road, Camperdown, NSW 2050, Australia) b School of Biomedical Sciences C42, University of Sydney, East Street, Lidcombe, NSW 2141,Australia (e-mail: hdag6201@mail, usyd. edu.au) Although cisplatin is a widely used anticancer drug, it has a number of side-effects and also cancer cells develop resistance to the continued use of cisplatin. In attempt to reduce the side-effects and to widen the spectrum of activity, thousands of different platinum complexes have been prepared and tested. Since it is found that all cisplatin analogues in general have a similar spectrum of activity, it is logical to assume that in order to have a different spectrum of activity the designed complexes should have a structure different from that of cisplatin such that they may not obey the classical structure activity relationship. We have designed a number of heteronuclear metal complexes, some of which have shown activity against a number of ovarian and other cancer cell-lines. This poster describes the activities of the designed complexes and the nature of their interaction with DNA.
Interaction of lanthanide-DO3A complexes with adjacent glutamate carboxylate groups in Calbindin D9K Nicola D'Amelio bx, Silvio Aimea, Ivano Bertinib, Marco Fragaib, Yong-Min Lee b, Claudio Luchinat d and Gianni Valensin ° a Department b Department Italy CDepartment d Department
of Chemistry, I.F.M., University of Torino, via P. Giuria, 7, 1-10125, Torino, Italy of Chemistry, University of Florence, via L. Sacconi, 6, 50019, Sesto Fiorentino, of Chemistry, University of Siena, Via A. Moro, 53100, Siena, Italy of Soil Science and Plant Nutrition, University of Florence, Florence, Italy
The paramagnetic properties of lanthanides can be exploited for getting insight in specific parts of protein surface. Due to the high affinity of lanthanides for oxygen donors, carboxylate groups can be used as specific targets for the interaction. The DO3A ligand is particularly useful in these studies, for it coordinates lanthanides leaving two sites available for other donors. A solution of a 15N labeled sample protein, Calbindin D9K (75 residues) was titrated up to 200% with Gd3+-DO3A complex, and a combination of ~SN-ZHHSQC with inversion recovery experiment was used for measuring paramagnetic contributions to longitudinal relaxation rates after each addition. Relaxation data were used as distance constraints with different assumptions on the number of interacting complexes and their occupancies and the presence of four preferential interaction sites on the protein surface was inferred. Here we demonstrate that Gd(III)DO3A complex does interact with exposed carboxylates of Glu and Asp side chains. The interaction is apparently selective for pairs of close carboxylates gathered in one region thus providing an easy and quick tool for obtaining insight into the structural features of the protein.
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Fur, an iron metalloregulatory protein from bacteria; reactivity with nitric oxide c
Beno~t d ' A u t r 6 a u x a, O l i v i e r H o m e r a, C l a u d i n e J e a n d e y a, J e a n - L o u i s O d d o u a, B e a t e B e r s c h , J e a n M a r c L a t o u r a, D a n i 6 l e T o u a t i b, I s a b e l l e M i c h a u d - S o r e t a. t/
S e r v i c e de Chimie I n o r g a n i q u e et Biologique, D R F M C / C E A - G r e n o b l e , 17 rue des Martyrs, 38054, G r e n o b l e cedex09, France. E m a i l ." d a u t r e a u @ d r f m c . c e n g . c e a . f r . b L a b o r a t o i r e de G~nOtique MolOculaire des R @ o n s e s Adaptatives, Institut J a c q u e s Monod, 2 p l a c e Jussieu, 75251, P a r i s cedex05, France. c L a b o r a t o i r e de R M N des Protdines, Institut de B i o l o g i e Structurale, 41 rue Jules Horowitz, 38042, Grenoble cedex09, France. The Fur protein is involved in the regulation of genes related to iron uptake in Gram-negative bacteria such as E. coli Fur is a dimer which has been proposed to bind iron as corepressor and then to act as a negative regulator via sequencespecific protein-DNA interactions at the promoter regions of iron regulated genes. The purified Fur protein is able to bind one metal dication (Co 2+, Mn 2+ and Fe 2÷) per monomer and also contains a tightly cysteines-bound zinc per monomer. Recently, Fur has been proposed, from in vivo experiments, to be inactivated by NO donors allowing then the expression of the flavohemoglobin gene which is under Fur control in Salmonella typhimurium 0). The interaction of NO and the Fur protein from E. coli has been investigated. In vivo and in vitro assays of the Fur activity in presence of NO together with the spectroscopic characterization of the iron site in absence (z) or in presence of nitric oxide will be present here. !~ 2,
Crawford, M.J.; Goldberg, D.E.; J. Biol Chem. 1998, 273, 51, 34028-34032 Jacquamet, L.; Dole, F.; Jeandey, C.; Oddou, J. L.; Perret, E.; Le Pape, L.; Aberdam, D.; Hazemann, J. L.; MichaudSoret, I.; Latour, J. M. J. Am. Chem. Soc. 2000, 122, 394-395.
Two blue copper proteins and their interaction with lipids and redox partners Eva Danielsena, Lars ElkjaerJorgensena " D e p a r t m e n t o f M a t h e m a t i c s and Physics, The R o y a l Veterinary and A g r i c u l t u r a l University, T h o r v a l d s e n s v e j 40, DK-1871 F r e d e r i k s b e r g C, D e n m a r k (e-mail. e l k j a e r @ k v l . d k and eda@kvl, dk) Cupredoxins can be encountered in several places in biology. It is a class of smaller (about 10-15 kD) globular [3barrel proteins containing a type I copper site. As part of their function in electron transfer, they form short-lived interaction complexes with their redox parmers. This is a study of the interaction of plastocyanin and amicyanin with their redox partners as well as with lipid membranes. The two cupredoxins have been picked since they function in similar environments (plastocyanin in the thylakoid lumen of plants and amicyanin in the periplasmic space of bacteria) allowing for a comparative study. The poster will contain binding studies between well-characterised lipid vesicles of DMPC and DGDG and proteins where the copper has been substituted with either radioactive cadmium or silver. Measurements include radioactive labeling, and Perturbed Angular Correlations (PAC)-spectroscopy for varying pH and redox states. The PAC experiments can measure the structure and binding of interaction complexes between metal-substituted copper proteins and their redox partner for varying pH, redox state, and ionic strength. Investigations were made on amicyanin+MADH, plastocyanin+PS 1 ~, and plastocyanin+cytochrome b6f. Some results include Observation of Ag+-plastocyanin binding to lipids at low pH Derivation of a dissociation constant of approximately 50 gM for the Cd2+-amicyanin+MADH complex Observation of two structural species of free amicyanin with different pH dependency 1.
Danielsen E. et al, Biochemistry, 38, 11531 - 11540 (1999)
The Research Training Network in the fifth programme of the European Commission, and the Danish Technical Research Council are acknowledged for financial support.
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Comparative Studies on Different Forms of Human Phenylalanine Hydroxylase
Sylvia Daoud Kinziea, Mui Sama, and Mahdi Abu-Omara ~Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Avenue, 90095, Los Angeles, California, U.S.A. (email: [email protected]) Human phenylalanine hydroxylase (hPAH) is a non-heme iron-dependent enzyme that catalyzes the hydroxylation of L-Phe into L-Tyr in the presence of tetrahydrobiopterin and molecular oxygen. Single base mutations or small deletions in the PAH gene result in phenylketonuria (PKU), an autosomal disorder of phenylalanine metabolism. In the present work, we have expressed and characterized recombinant hPAH in Escherichia coli using the pMAL and the thioredoxin expression systems. The wild-type form and three different deletion mutants (Thr117-Ser411, Thr117-Leu424, Thr117Lys452) were expressed with high specific activity. Each form revealed a different state of oligomerization as determined by gel filtration chromatography. The wild-type protein consists of active dimeric and tetrameric forms while the 117-411 is monomeric, the 117o424 dirneric and the 117-452 tetrameric. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) techniques to monitor folding and unfolding transitions reveal differences in the stability of the different forms. The melting temperature for the wild-type tetramer is 52 °C + 2 °C and that of the deletion mutants is 60 °C + 2 °C. However, no additional stabilization is seen in the presence of iron(II) which is absolutely required for catalytic activity. To further understand the structural stability and folding properties of the protein, we are examining the catalytic, folding and oligomerization defects of the PAH protein caused by several PKU and non-PKU mutations. The March of Dirnes Foundation is acknowledged for financial support.
Preferential formation of a 1,4-interstrand DNA crosslink rather than a 1,2intrastrand crosslink by a dinuclear platinum complex
Murray S. Daviesa, Susan J. Bemers-Pricea, John W. Coxb, Donald S. Thomasa, Nicholas Farrellb aDepartment of Chemistry, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, AUSTRALIA. (email: [email protected], edu.au) bDepartment of Chemistry, Virginia Commonwealth University, 1001 Main Street, Richmond, Virginia, 23284-2006, USA. Competition between formation of a 1,4-GG interstrand or a 1,2-GG intrastrand crosslink (in both 3',3" and 5',5' directions) has been examined by following the reaction between 15N-[{transPtCI(NH3)2} z(~t-NH2(CHz)6NH2)] 2+, ( 1,1/t,t (n=6)), and the duplex 5 'd(ATACATGGTACATA)-3".5"-d(TATGTACCATGTAT)-3" (I) by [~H,tSN] HSQC NMR spectroscopy. The reaction affords one major monofunctional adduct and two bifunctional adduct conformers. Analysis of the NOESY spectrum of the final product % confirmed the presence of two conformers of the 1,4-GG interstrand cross-link in the 5',5' direction. Monofunctional binding of the drug is considerably faster with (I) than with the 12 mer duplex, l 5"-{d(ATATGTACATAT)}2, but the rate of closure of the monofunctional adduct to form the bifunctional crosslink is similar. 1. Cox, J. W., Berners-Price, S. J., Davies, M. S., Qu, Y. and Farrell, N., J. Am. Chem. Soc., 123, 1316-1326 (2001). The authors thank the Australian Research Council, the U. S. National Institutes of Health, U. S. National Science Foundation and the American Cancer Society for t'mancial support.
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Evidence of c o m p o u n d I and compound II ferryl species from the reaction of hemoglobin I from lucina pectinata with hydrogen peroxide Walleska De Jesfis-Bonilla b, Jos6 Cerda a, Juan L6pez-Garriga b, Eunice Ramirez-Mel6ndezb Department of Chemistry, Michigan State University, East Lansing, Michigan, USA b Department o f Chemisty, University of Puerto Rico, Mayagiiez Campus, P.O. Box 9019, 00681-9019, Mayagiiez, Puerto Rico (e-mail. wdejesus@sonw, uprm.edu) The formation of ferryl heme species has been identified as the main intermediates in heme protein peroxidative reactions.l'2 We report UV-Vis and stopped-flow kinetic measurements, which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce the ferryl species compound I and compound II. Compound I appears to be relatively stable displaying an absorption band at 648 nm. The rate constant value (k4t) for the oxidation of the ferrous HbI is 25 M l s 1, nearly four times smaller than the reported for myoglobin; the conversion of compound I to compound II (k23) is 3.0 x 10-2s1, more than a hundred times smaller than myoglobin; and the oxidation of the ferric heme (k12+k13) is 2.0 x 102 Mts -1. The k23/k4! ratio shows that HbI has a higher peroxidative capacity for removing H202 from solution in relation to the ferrous oxidation. In HbI from L. pectinata the stabilization of the ferryl species is due to the unusual collection of amino acid residues (Q64, F29, F43, F68) in the heme pocket active site of the protein. Resonance Raman studies confirm that the compound II ferry1 vibration frequency is present at 805 cm -1 for the HbIFeW=160, and at 769 cm-1 for the HbIFeIV=lSO species. This higher ferryl vibrational mode result implies the lack of hydrogen bonding between the Q64 and the ferryl species. This unusual vibrational mode also suggests that the carbonyl group of Q64 in HbI points toward the iron of the heme group, increasing the electron density in the heme pocket moiety. 1. 2.
Snyder S., Welty E., Walder R., William L. and Walder J., Proc. Natl. Acad. Sci. USA, 84, 7280-7284 (1987). Alayash A.I., Ryan B. A., Eich R.F., Olson J.S. and Cashon R.E., J. Biol. Chem., 274, 2029-2037 (1999).
This project was supported, in part, by NSF (MCB-9974961) and NIH (MBRS/SCORE-SO6GM08103-27).
Electron transfer between proteins: effect of cross-linking Thvra E. de Jongh,a Irene M.C. van Amsterdam,a Marcellus Ubbink,a Oliver Einsle,b Albrecht Messerschmidt, b Angelo Merli, c Davide Cavazzinif Gian Luigi Rossi ~ and Gerard W. Canters ~ a Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, the Netherlands b Max-Planck-Institutffir Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, 82152 Martinsried, Germany c Instituto di Scienze Biochimiche, Universita d e g l i s t u d i di P arm a, Parco della Scienze 23A, 143100 Parma, Italy In order to gain more insight into the relative orientation of proteins in short-lived complexes of redox proteins we have analysed the effect of cross-linking on the electron transfer between two molecules of the blue copper protein azurin, in the so-called electron self-exchange (e.s.e.) reaction. In the crystal packing of wild type azurin two molecules face each other with opposing hydrophobic patches, which is thought to be similar to the transient complex that is formed during the e.s.e, reaction in solution. The Asn42 residues of each monomer are in close proximity, which makes them, for the purpose of cross-linking, suitable for mutagenesis. Asn42 was replaced by cysteine and two different dimers were constructed: 1) a disulfide bridged dimer (Cys42-S-S-Cys42) and 0 0 2) a 'BMME-dimer', which is cross-linked using the bifunctional symmetric linker bismaleimidemethylether (BMME) creating a Cys42-S-BMME-S-Cys42 linkage. The The cross-linking electron transfer rates within, as well as between, the dimers were analysed by NMR. The results were complemented by the crystallographic data obtained of these constructs. reagent bis-
~ -CH~-O-CH~-N~
maleimidemethylether
198
Journal of Inorganic Biochemistry 86 (2001)
Mutagenesis of the cytochrome c peroxidase from Rhodobacter capsulatus L i n a D e S m e t a, G r a h a m W. P e t t i g r e w b, Y v e s G u i s e z a a n d J o z e f V a n B e e u m e n a
a Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Ledeganckstr. 35, 9000, Gent, Belgium, b Department of Preclinical Veterinary studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall Edinburgh EH9 1QH, UK Cytochrome c peroxidases (CCP) have been found in yeast and bacteria. In bacteria two systems are available for the scavenging of potential harmful hydrogen peroxide: cytoplasmic catalase and periplasmic dihaem CCP. The latter catalyzes the oxidation of ferrocytochrome c and the reduction of H202. The reaction resembles that carried out by the haem b containing CCP from yeast, except that the BCCP's do not create a free radical during catalysis but, instead, use two haem c groups. These haems have totally different properties, one haem is low-potential and acts as the peroxidatic center, while the other is high potential and functions as the electron transfering haem. We were able to overproduce the Rb. capsulatus CCP in E. coli. The recombinant protein was purified in a four-step procedure. Diffraction-quality crystals were obtained under two conditions. The crystal structure of the dihaem cytochrome c peroxidase was determined by molecular replacement using Ps. aeruginosa as a model 1. The structure solved is that of the completely oxidized enzyme. In this form the N-terminal haem iron is ligated with histidines 55 and 74 and therefore the low potential haem, while the C-terminal haem c is ligated by histidine 205 and methionine 278. The completely oxidized enzyme is inactive. The reaction mechanism of the CCP's is rather complex and involves changes in iron ligands and spin states. The activity of the enzyme is also calcium dependent 2. Two possible models for hydrogen peroxide reduction by CCP have been proposed so far ~.3. In the first model a switch to a high spin state at the low potential N-terminal haem is the main event. In the second model both a spin switch and a ligand switch occurs. The N-terminal haem becomes the high-potential electron transferring haem, while the C-terminal haem becomes a penta-coordinate peroxidatic center. Mutagenesis was used to give more insight in the reaction cycle. We will present the structure and characterisation of the wild type enzyme and of several mutants. 1.
Fulop V.et al., Structure, 3, 1225-1233 (1995); 2. Gilmour R. et al., Biochem. J,. 300, 907-914, (1994); 3. Prazeres S. et al., J. Biol. Chem., 31, 24264-24269 (1995)
Does stereospecific oxidation by compound I of cytochrome P450 ever proceed in a concerted manner? Sam P. de Visser. Francois Ogliaro and Sason Shaik Department of Organic Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Density Functional B3LYP/LACVP calculations were performed on a Cytochrome P450 model Compound I, oxoiron porphyrin thiol [FeOPorSH]. The reaction path for the epoxidation of ethene by Compound I was studied, v3 It was found that the reaction proceeds stepwise via a radical intermediate, which can exist either in a low spin (doublet) or high spin (quartet) form. Additionally, the radical intermediate can have the iron in oxidation states Fem or Fe w. The formation of the radical complexes from reactants was found to proceed via barriers of 13.9 kcal mol 1 (4FelV) and 14.9 kcal mol l (2FelV). Both low spin radical complexes react in a barrierless fashion to give an epoxide complex. On the high spin surface small barriers of 2.3 kcal mol l (Fe TM) and 7.2 kcal mol l (Fe ul) leading to an epoxidation complex were obtained. In addition, we have calculated the barrier for synchronous attack of the ethene leading to the epoxide complex in a concerted manner. This barrier was found to be 21.1 kcal mo1-1. Consequently, the concerted reaction path is not available for this process. Therefore, the stereospecifity of P450 oxidation originates from the asynchronous mechanism in the doublet spin state whereas occasional stereochemistry and side products originate from the stepwise mechanism in the quartet spin state. I. 2. 3.
S.P. de Visser, F. Ogliaro, N. Harris and S. Shaik J. Am. Chem. Soc. 2001, 123, 3037. S.P. de Visser, F. Ogliaro and S. Shaik Angew. Chem. Int. Ed. 2001, in press. S.P. de Visser, F. Ogliaro and S. Shaik submittedfor publication.
Journal of Inorganic Biochemistry 86 (2001)
199
Biochemical characterization of the reductases involved in denitrification in the hyperthermophilic pyrobaculum aerophilum and purification of the NO reductase S. de V r i e s ", M . J . F . S t r a m p r a a d a, I. S c h r 6 d e r b
aKluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands bDepartment of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095-1489 The molecular biology and biochemistry of denitrification in Proteobacteria has been studied extensively (1). However, little is known about denitrification in Archaea. Pyrobaculum aerophilum is a hyperthermophilic Archaeon growing optimally at temperatures of 100°C utilizing nitrate or oxygen as terminal electron acceptors for heterotrophic growth. Previous work has indicated that nitrate is reduced via nitrite, NO and N20 to N2, i.e. in both Proteobacteria and Archaea the denitrification intermediates are the same. Our aim is to determine the structure and function of the four reductases NAR, NIR, NOR and NzOR involved in denitrification in P. aerophilum. To this end, we have developed both polarographic and colorimetric assays to determine the activities of the four reductases continuously up to temperatures of 95 °C enabling the rapid detection of active fractions obtained during purification. Enzyme assays with whole membranes using menahydroquinone (MQH2) as electron donor (the natural quinone in P. aerophilum is a (novel type of) menaquinone) with nitrate, nitrite, nitric oxide or nitrous oxide as acceptor showed that in P. aerophilum all four reductases are membrane bound, in contrast to Proteobacteria where NIR and N2OR are watersoluble enzymes residing in the periplasmic space together with small cytochromes and cupredoxins. P. aerophilum does not contain soluble c-cytochromes or cupredoxins. NOR was extracted with laurlymaltoside and after CM-sepharose- and HAP- chromatography purified to homogeneity. The purified NOR from P. aerophilum is single subunit enzyme displaying high MQH2: NO oxidoreductase activty upto 95 °C. The enzyme contains heme and non-heme iron in the ratio of 2:1. Using EPR and UV-Vis spectroscopy we showed that one of the hemes is a low-spin heme b like heme (gz= 3.015; gy= 2.226; g×= 1.45), the other is high-spin becoming low spin upon binding of CO.
Conformational transitions of polyAx2polyU in the presence of Mg2+ in solutions with different ionic strength. M.V. Dcgtyar, V.A. Sorokin, G.O. Gladchenko, V.A. Valeev, Yu.P. Blagoi B I. Verkin Institute for Low Temperature Physics and Engineering, National Academy of Sciences of Ukraine,Lenin ave. 47, 61164, Kharkov, Ukraine, (e-mail. my [email protected]) Heat induced conformational transitions of polyAx2polyU (Ax2U) in the presence of Mg 2÷ were studied by UV spectroscopy at solutions with different ionic strength: 10-~, 3x10 -2 and 10~M Na% At low ionic strength (10 .2 and 3x 10-ZM Na ÷) two transitions take place: a) Ax2U ~ AxU + polyU, b) AxU --, polyA + polyU. At 10-tM Na ÷ Ax2U dissociates directly to one stranded species. Full faze diagrams of conformational equilibrium in polynucleotides solutions are obtained. Increase of [Mg 2+] results in the increase of thermostability of all helix structures. Concentrational dependencies of 3--*2, 2--+1 and 3-~1 melting temperatures on [Mg 2+] permit us to calculate Mg 2+ binding constants using equilibrium binding theory [1]. At low binding degree the values of association constants (table 1) agree to the extent of 10% with data from [2] recalculated for .10.2 and 3 x 10.2 M Na + using slope
OlgK
_ -1.6.
0 ig[Na + ] Table 1. Binding constants of Mg 2+ interacting with Ax2U and AxU (K, M l). [Na +],M 10.2 3 x 10z 101 our data [2] our data [2] our data Ax2U 9x104 8x103 2x103 AxU
8x104
8.5×10 4
10 4
1.3x104
1 Lazurkin Yu.S., Frank-Kamenetskii M.D., Trifonov E.N. Biopolymers, 2, 1253 - 1306 (1970) 2. Ohyama T., Cowan I.A. JBJC, 1, 83-89 (1996)
Journal of Inorganic Biochemistry 86 (2001)
200
COME, the ontology for bioinorganic centres in proteins Kirill D e g t y a r e n k o , S e r g i o C o n t r i n o
EMBL Outstation, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CBIO 1SD, U.K. (e-mail. [email protected], uk) Annotation and interoperability of biological databases in the "post-genornic" era is seriously hampered by the lack of a common nomenclature for biochemical entities. The Gene Ontology Consortium I addresses the problem by developing ontologies for biological process, molecular function and cellular component. It is hoped that Gene Ontology (GO) will become the lingua franca for annotation of biological databases. However, the bioinformatics community has paid too little attention to the complex biochemical structures such as metalloproteins 2 and it also remains for the wealth of knowledge accumulated by bioinorganic chemistry to be organised in a systematic way. COME (Co-Ordination of Metals) represents the ontology for bioinorganic centres in complex proteins. COME consists of three types of entities: molecule (MOL), bioinorganic motif (BIM), and bioinorganic proteins (PRX), each entity is assigned a unique identifier. MOL entity represents "small molecule" (as opposed to macromolecule) which, in complex with a polypeptide, forms a functional protein. BIM consists of at least one centre (metal atom, inorganic cluster, organic molecule) and two or more endogenous and/or exogenous ligands. The PRX entity refers to the functional protein as well as separate protein domains and suhunits. It may include MOL, BIM as well as other PRX entities. The data are currently stored in XML format and are available from the web site 3. The relational implementation of the database is under development. 1. 2. 3.
The Gene Ontology Consortium, Nature Genetics 25, 25-29 (2000) Degtyarenko, K.N., Bioinformatics, 16, 851-864 (2000) http://www.ebi.ac.uk/-kirill/come/
New myoglobin reconstituted with an iron porphycene as a structural isomer of heme moiety Hirohisa Dejima
a,
Takashi Hayashi
a,b, T a k a s h i
M a t s u o b, Y o s h i o H i s a e d a a
~Department of Chemistry and Biochemistry, Kyushu University and bTOREST in Japan Science and Technology Corporation, Fukuoka 812-8581, JAPAN (e-mail: d e j i i t c m @ m b o x . n c . k y u s h u - u . a c . j p ) Myoglobin (Mb) is an oxygen storage protein containing protoporphyrin IX iron complex as a prosthetic group. To change the physiological and/or chemical property of the myoglobin, we prepared an iron mesoporphycene as a structural isomer of mesoporphyrin and then inserted it into apomyoglobin by usual method as shown in the following scheme. The reconstituted metMb exhibits a characteristic reconstitution absorption spectrum with peaks at 387 (Soret), 564, and 624 nm. ESI-TOF-MS spectrum of the reconstituted metMb shows two peaks at ngz = 17,577 and 16,959, corresponding to holoMb and apoMb, respectively. After the reduction of the metMb to deoxyMb, we monitered CO complex at 393 HOOC COOH (Soret) and 613 nm in pH 7.0, 100 mM phosphate buffer apomyoglobin iron porphycene involving CO gas. Here, we wish to report the characterization and property of the reconstituted Mb with the iron porphycene.