FLAVINSAND FLAVOPROTEINS

1999

PROCEEDINGS OF THE THIRTEENTH

INTERNATIONAL SYMPOSIUM

KONSTANZ, GERMANY, AUGUST 29 - SEPTEMBER 4, 1999

EDITORS

S. GHISLA • P. KRONECK

P. MACHEROUX • H. SUND

RUDOLF WEBER

AGENCY FOR SCIENTIFIC PUBLICATIONSBERLIN 1999

XV

Contents

I. General Aspects:History,Chemistry, Physics and Theory

Living through various phases of flavin research 3Helmut Beinert

Synthetic Models of flavoenzyme activity 17Vincent Rotello

NMR-studies of flavocytochrome b2 reconstituted with15N, I3C labelled flavinGarit Fleischmann, Franz Miiller, Heinz Riiterjans, Florence Lederer

One and two electron redox cycles in flavin-dependent dehydrations 31W. Buckel, I. Cinkaya, S. Dickert, U.Eikmanns, A. Gerhardt, M. Hans,M. Liesert, W. Tammer, A. J. Pierik, E. F. Pai

Dipole moments and polarizabilities of flavins explored usingStark-effect spectroscopy ' 41Robert J. Stanley, Haishan Jang

Flavin binding thermodynamics in Enterobacter cloacaenitroreductase 45Ronald L. Koder, Michael E. Rodgers, Anne-Frances Miller

Regulation of flavin functions by hydrogen bondings 49Yumihiko Yano, Takeshi Kajiki, Hideki Moriya

The hydrogen bonding in flavoproteinThe effect of hydrogen bonding of flavin (neutral semiquinone state) 53Yoshitaka Watanabe

Substituent effect on redox states, spin densities and hyperfinecouplings of free flavins and their 5-deaza analogues 59Ryszard Zielinski. Henryk Szymusiak

Theoretical destabilization of the flavin semiquinone of Enterobactercloacae nitroreductase by a hydrogen-bonding -bending mechanism 63Joseph D. Walsh, Anne-Frances Miller

XVI

Supramolecular models of flavoenzyme redox processes 67Catherine Mclntosh, Angelika Niemz, Vincent Rotello

Electronic effects of 7 and 8 ring substituents as predictors offlavin oxidation-reduction potentials 71Dale E. Edmondson, Sandro Ghisla

Autoxidation of photoreduced 3,4-dihydro-6.7-dimethyl-3-oxo-4-D-ribityl-2-quinoxalinecarboxamide, an analog of riboflavin,and identification of oxygenated intermediates 77K. Matsui, Y. Nishina, K. Sato, K. Shiga

Intermediate products of 1,5-dihydroflavins autoxidationon the basis of density functional theory studies 81Henryk Szymusiak, Ryszard Zielinski

II. Spectroscopy

Advanced EPR spectroscopic studies of mutants of ferredoxin-NADP+ reductase from pea 87Milagros Medina, Carlos Gomez-Moreno, Richard Cammack,Adrian K. Arakaki, Nestor Carillo, Eduardo A. Ceccarelli

Radical intermediates in Escherichia coli DNA photolyase:EPR and ENDOR studies 91Gerald Richter, Christopher W. M. Kay, K. Struck,Peter Sadewater, Klaus Mobius, Stefan Weber

Resonance Raman study on the interaction between Hog kidneyD-amino acid oxidase and substrate analogs 95Ruiwen Shi, Yasuzo Nishina, Kyosuke Sato, Kiyoshi Shiga, Retsu Miura

Direct measurements of ultrafast excited state quenching ofisoalloxazine by adenine in FAD 99Robert J. Stanley, Alexander W. MacFarlane IV

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III. Flavins and Electrons

1. Electron-Transfer in Heme-containing Systems

Flavocytochrome c3: The structure and mechanism revealed 105S.K Chapman, C.A. Morrison, G.A. Reid, S.L. Pealing,P. Taylor, M.D. Walkinshaw

Two FCSD flavoproteins involved in sulfur metabolism and a thiosulfateutilization gene cluster from the green phototrophic bacterium,Chlorobium limicola (strain Tassajara) 115Fabienne Verte, Yves Guisez, Terrance E. Meyer, Michael A.CusanovichJozef J. Van Beeumen

A flavohemoprotein from the cellulolytic fungus Humicola insolvenscontains 6-hydroxy-FAD as the dominant active cofactor 119Kiyohiko Igarashi, Marc F. J. M. Verhagen, Masahiro Samejima,Martin Schiilein, Karl-Erik L. Erikson, Takeshi Nishino

Changing the heme ligands of flavocytochrome b2:site-directed mutagenesis of histidine 66 to cysteine 123Christopher G. Mowat, Stephen K. Chapman,Caroline S. Miles, Graeme A. Reid, A.W. Munro

Cloning and characterization of a membrane-bound flavocytochrome cfrom the purple phototrophic sulfur bacteriumEctothiorhodospira vacuolata 127Vesna Kostanjevecki, Yves Guisez, Terrance E. Meyer,Michael A. Cusanovich, Jozef J. Van Beeumen

Electron transfer from FAD to heme-Fe in plant NADH:nitrate reductase 131Jeffrey A. Mertens, Wilbur H. Campbell, Lawrie Skipper, David J. Lowe

Expression, purification and mutagenesis of flavocytochrome c-sulfidedehydrogenase from Chromatium rinosum 135Lina De Smet, Yves Guisez,Terrance E. Meyer, Michael A. Cusanovich,Jozef J. Van Beeumen

The active site base of the fumarate reductase fromShewanellaftigidimarina 139Mary K. Doherty, Stephen K. ChapmanCaroline S. Miles, Graeme A. Reid

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Molecular recognition in the flavin domain of flavocytochrome b2 143Ruth K. Moysey, F. Welsh, Stephen K. Chapman, S.L. Rivers,Graeme A. Reid

Key substrate binding residues in flavocytochrome P450 BM3 147T.W.B. Ost, M.A.Noble, S.K.Chapman, Caroline S. Miles, J. Murdoch,G.A. Reid, A.W. Munro

The flavoprotein domains of nitric oxide synthase isoforms:site of possible regulatory control of flavin to heme electron transfer 151R. Timothy Miller, Pavel Martasek, Jonathan S. Nishimura, Satya Panda,Dawn E. Harris, Linda J Roman, Bettie Sue Masters, Jung-Ja P. Kim

Analysis of the redox properties of the human diflavin enzyme NADPH-cytochrome P450 reductases and its domains 155Andrew W. Munro, T. McSorley, Michael A. Noble, Laura Robledo,Stephen K. Chapman

Crystallographic insights into the hydride transfer mechanism ofNADPH-cytochrome P450 oxidoreductase 159Paul A. Hubbard, Rosemary Paschke, Jung-Ja P. KimAnna L. Shen, Charles B. Kasper

Flavodoxin as module for transferring electrons to differentc-type and P450 cytochromes in artificial redox chains 163Sheila J. Sadeghi, Yergalem T. Meharenna and Gianfranco Gilardi

Mechanic studies on the one-electron reduction of quinonesby neuronal nitric-oxide reductase domain 167Hiroyuki Matsuda, Shigenobu Kimura and Takashi Iyanagi

Calmodulin activates intramolecular electron transfer betweenthe two flavins of neuronal nitric-oxide synthase reductase domain 171Hiroyuki Matsuda, Shigenobu Kimura, Takashi Iyanagi

2. Electron Transfer in Flavodoxins

The binding of FMN to Anabaena apoflavodoxin 175Anabel Lostao, Fatna Daoudi, J. Sancho

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Egression of recombinantflavodoxin from M^gasp/wera elsdem;effects of mutating the residues (M57, W91) that sandwich thednnethylisoalloxazine of the bound FMN 179Mary E. Gallagher, Susan M. Geoghegan, Stephen G. Mayhew

Role of conformational dynamics and associated electrostaticand hydrogen bonding interactions in the regulation ofredox potentials in the Clostridium beijerinckii flavodoxin 183Richard P. Swenson, Mumtaz Kasim, Luke H. Bradley, Larry Druhan

Effects of pH on the 13C and ISN NMR spectra of the hydroquinoneof Desulfovibrio vulgaris flavodoxin and its G61A mutant 187Garry N. Yalloway, Stephen G. Mayhew, Sjef J. Boeren, Jacques Vervoort

Studies on urea-unfolding of flavodoxin and apoflavodoxin fromDesulfovibrio vulgaris (Hildenborough) 191Brian 6 Nuallain, Stephen G. Mayhew

3. Various Aspects

Flavin-linked redox components required for AhpC reductionin alkyl hydroperoxide reductase systems . 195Leslie B. Poole

Overexpression of the bifunctional chorismate sythase ofNeurospora crassa 203Karina Kitzing, Nikolaus Amrhein, Peter Macheroux

Amino acid substitutions near the FAD in NADH-cytochrome bs

reductase: roles of Arg*3 and Thr6* in the electron transfer 207Shigenobu Kimura, Masanori Kawamura, Takashi Iyanagi

Comparison of the spectral properties between pigMegasphaera elsdenii electron-transferring flavoproteins 211Kyosuke Sato, Yasuzo Nishina, Kiyoshi Shiga

Solvent isotope effects on electron transfer in xanthine oxidase 215Russ Hill, Robert F. Anderson

The family of A-type flavoproteins: new members and definitionof unique sequence fingerprints 219Claudio M. Gomes, Miguel Teixeira, Alain Wasserfallen

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Formation of ETF hydroquinone when complexed totrimethylamine dehydrogenase 223Mei-Huei Jang, Russ Hille, Nigel S. Scrutton

Evidence that Megasphaera elsdenii synthesises two differentelectron-transferring flavoproteins 227Zuhair Nasrallah, Hugh O'Neill, Stephen G. Mayhew

IV. 3D-Structures of Flavoproteins

Structural flavinology on the brink 233P. Andrew Karplus

Crystal structures of ,,unactivated" p-hydroxybenzoate hydroxylase 239Michel H. M. Eppink, Willem J. H. van Berkel, Alex Tepliakov,Herman A. Schreuder

NMR-studies on FMN-binding protein fromDesulfovibrio vulgaris (Miyazaki F) 243Masaya Kitamura, Hideo Inoue, Edwards Liepinsh, Gottfried Otting

Crystal structure of the respiratory fumarate reductase of reductaseof Shewanella putrefaciens MR-1:member of a novel flavocytochrome family 247David Leys, Terrance E. Meyer , Alexandre I. Tsapin,Michael A. Cusanovich, Jozef J. Van Beeumen

The domain structure of flavocytochrome c3 fromShewanella frigidiinarina 251S.L. Pealing, S.K. Chapman, P. Taylor, G.A. Reid, M.D. Walkinshaw

Structural biology of L-aspartate oxidase and polyamine oxidase 255Claudia Binda, Andrea Mattevi, G. Tedeschi, A. Negri,Rodolfo Federico, Riccardo Angelini

A new functional model for the Escherichia coli sulfite reductase:the aipi complex 259Jaques Coves, Mahel Zeghouf, Marc Fontecave

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V. Flavins and Pyridine Nucleotides

Structural and functional properties of corn rootferredoxin-NADP+-reductase 265Alessandro Aliverti, Cristian Ferioli, Monica Spinola, Debora Raimondi,GiulianaZanetti, Casey Finnerty, RickFaber, P. Andrew Karplus

Role of the C-terminal tyrosine of plant ferredoxin-NADP+-reductasein NADP+ binding affinity and pyridine nucleotide specificity 269LucianoPiubelli, Alessandro Aliverti, GiulianaZanetti, Adrian K Arakaki,Nestor Carrillo, Eduardo A Ceccarelli, P. Andrew Karplus

Role of glutamic acid 139 in the catalytic mechanism offerredoxin-NADP+-reductase from Anabaena PCC7119 273Merche Faro, Milagros Medina, Carlos G6mez-Moreno,John K. Hurley, T.B. Brodi, Gordon Tollin

Characterisation of flavodoxin (ferredoxin) NADP+ oxidoreductase andflavodoxin; key components of electron transfer in Escherichia coli 277Lisa Mclver, Claire Leadbeater, Dominic J. Campopiano, Robert L. Baxter,Stephen K. Chapman and Andrew W. Munro

Sequence analysis and overexpression of putative NADH oxidasesfrom the hyperthermophilic archeaons Sulfolobus solfataricus andPyrococcus horikoshii 281Edward J. Crane III, Donald E. Ward, John van der Oost,Qunxin She, Roger Garrett

On new artificial mediator accepting NAD(P)H oxidoreductases fromClostridium thermoaceticum and Clostridiumformicoaceticum 285Helmut Giinther, Katrin Walter, Richard Feicht, Peter Kohler, Helmut Simon

Flavin reductase Fre from Escherichia coli: identification ofreaction intermediates and evidence for a new mode of bindingfor reduced pyrSdine nucleotides 289Vincent Niviere, Marc Fontecave, Maria A. Vanoni, Franck Fieschi

Determinants of cofactor specificity in Anabaena PCC7119ferredoxin-NADP+-reductase 293Jesus Tejero, Alejandra Luquita, Koert Grever, Carlos G6mez-Moreno,Milagros Medina, R. Perham

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VI. Flavins and Light

1. Photochemistry, Blue Light Effects and Photolyases

Phototropin (nphl), a photoreceptor for phototropism, is anFMN-binding chromoprotein 299Winslow R. Briggs, John M. Christie, E. Knieb, Michael Salomon

DNA photolyase and cryptochrome 309Takeshi Todo

DCRY: A Drosophila photoreceptor protein implicated inlight entrainment of circadian rhythm 317Tomoko Ishiwaka, Takeshi Todo, Akira Matsumoto,Teiichi Tanimura, Shin Togashi, Ryu Ueda

Isolation and characterization of DNA photolyase/cytochromefamily in Zebra fish 321Yuri Kobayashi, Tomoko Ishikawa, Takeshi Todo,Hiromi Daiyasu, Hiroyuki Toh

2. Luciferase

Mechanism of reduced flavin transfer and complex formation betweenVibrio harveyi NADPH-FMN oxidoreductase and luciferase 325Shiao-Chun Tu, Benfang Lei, Mengyao Liu, Chih-Kwang Tang,Christopher Jeffers

Identification of P-flavin binding protein, and the genes forcobalamin-dependent methionine synthase andflavodoxin 1 in Vibrio flscheri 333Sabu Kasai

Modeling the intermediate IV of the luciferase reaction:characterization of the complex of 5-decylFMN-4a-OHwith Vibrio harveyi luciferase 337Benfang Lei, Qizhu Ding, Shiao-Chun Tu

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VII. Flavins and Oxygen

1. General Aspects

Coenzyme recognition by flavoprotein aromatic hydroxylases 343Willem J. H. van Berkel, Michel H. M. Eppink, Herman A. Schreuder

Wavin' flavins and passwords: dynamics and control in the reactionsof p-hydroxybehzoate hydroxylase 351Bruce A. Palfey, Kendra King Frederick, Rajit Basu, Dong XuDavid P. Ballou, Vincent Massey

2. Monooxygenases

Hydroxylation by flavin enzymes: evidence for NIH-shift mechanism 359Wolfgang Eisenreich, Claus Hultschig, Steffen Hartmann, Georg Fuchs,Adelbert Bacher, Sandro Ghisla

The intermediates involved in the catalytic reaction ofcyclohexanone monooxygenase 367Dawei Sheng, DavidP. BaHou, Vincent Massey

Catalytic properties of 2-hydroxybiphenyl 3-monooxygenase 371Winfried A. Suske, Hans-Peter Kohler, Willem J. H. van Berkel

Purification and some properties of acetophenone monooxygenase 375Marielle J.H. Moonen, Ivonne M.C.M. Rietjens, Willem J.H. van Berkel

Substrate and flavin activation in the hydroxylation catalyzed by/;-hydroxybenzoate hydroxylase: studies of the Lys297Met,Asn300Asp and Tyr385Phe forms reconstituted with 8rCl-FAD 379Mariliz Ortiz-Maldonado, Sara Aeschliman, David P. Ballou,Vincent Massey

Heterologous expression and kinetic characterization of humansqualene monooxygenase 383Brian P. Laden, Todd D. Porter

Novel two-component phenol hydroxylase from athermophilic Bacillus strain 387Ulrike Kirchner, R. Miiller, Willem J.H. van Berkel

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3. Various Aspects

Is charge-transfer complex formation essential for reductionof p-hydroxybenzoate hydroxylase? 391Barrie Entsch, Mariliz Ortiz-Maldonado, David P. Ballou

Single step analysis of catalysis of pyruvate oxidase fromLactobacillus plantarum. Kinetics, mechanism and regulation 395Kai Tittmann, Ralph Golbik, Gerhard Hiibner, Sandro Ghisla

Studies on the peroxide-reducing system of Thermits aquaticus. 401Catriona Logan, Stephen G. Mayhew

Converting a dehydrogenase into an oxidase 405Graeme A. Reid, Lars H. 0stergaard, Martin L. Goble, R. Moysey,Stephen K. Chapman

What protein features makep-hydroxybenzoate hydroxylasereact rapidly with oxygen? 409Willem J.H. van Berkel, Michel H.M. Eppink, Herman A. Schreuder,Mariliz Ortiz-Maldonado, Bruce A. Palfey, David P. Ballou, Barrie Entsch

VIII. Flavoproteins and MC=H-substrates"

1. A mine Dehydrogenation

Biochemical and structural characterization ofmonomeric sarcosine oxidase 415M.S. Jorns, M.A. Wagner, P. Trickey, F.S. Mathews

Substrate inhibition in trimethylamine dehydrogenase 423P. Roberts, J. Basran, E.K. Wilson, N.S. Scrutton, R. Hille

A novel heterotrimeric flavoprotein involved inbacterial nicotine catabolism 427Susann Schenk, Andre Hoelz, Karl Decker

High-level expression, structural, kinetic, and redox characterizationof recombinant human liver monoamine oxidase B 431Paige Newton-Vinson, Dale E. Edmondson

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A mechanism for monoamine oxidase involving a redox-active disulfide 435Rona R. Ramsay

Involvement of mitochondria! matrix in the holoenzyme formationof dimethylglycine dehydrogenase 439Carmen Brizio, Ernesto Quagliariello, Maria Barile, Salvatore Passarella,Annegret Otto,, Roderich Brandsch

Hydrogen tunnelling in amine dehydrogenases frommethylotrophic bacteria 443Jaswir Basran, Peter Roberts, Nigel S. Scruttdn, Michael J. Sutcliffe

Investigating the mechanism of C-H bond breakage inheterotetrameric sarcosine oxidase from Arthrobacter sp. 1-IN 447RJ. Harris, N.S. Scrutton, M.J. Sutcliffe, R. Meskys

The reductive half-reaction of trimethylamine dehydrogenasewith trimethylamine 451Mei-Huei Jang, Russ Hille, Jaswir Basran, Nigel S. Scrutton

Interaction of FAD analogues with the C406A mutant apoenzymeof human liver monoamine oxidase A 455Ravi K. Nandigama, Dale E. Edmondson

2. a-Hydroxy Acid Dehydrogenases

X-ray studies of recombinant rat kidney long-chain hydroxy acid oxidaseand of the recombinant flavin-binding domain ofbakers yeast flavocytochrome b2 459L.M. Cunane, J.D. Barton, Zhi-wei Chen, F.S. Mathews, A. Belmouden,K.H.D. Le\ F. Lederer, F.E. Welsh, S.K. Chapman, G.A: Reid

On the mechanistic value of the dehydrohalogenation reaction ofp-halogeno a-hydroxy acids catalysed by FMN-dependent hydroxyacid-oxidizing enzymes: a mutational analysis with flavocytochrome b2 463Sabrina Bodevin, Florence Lederer

D-Lactate dehydrogenase model. Mechanism of the oxidation ofmandelic acid by functionalized flavin mimics with metal ions 467Hideaki Ohshiro, Shin-ichi Kondo, Yumihiko Yano

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Mechanistic roles of the conserved residue arginine 277in (S)-mandelate dehydrogenase from Pseudomonas putida 471Isabelle E. Lehoux, Bharati Mitra

The effects of mutation of Asp 180 of L-lactate monooxygenase fromMycobacterium smegmatis. Reduction of three mutants by L-lactateand production of hydrogen peroxide 475Stephen A Sanders, Ute Miih, Vincent Massey, Charles H. Williams Jr

Reaction mechanism of L-lactate oxidase from Aerococcus viridans 479Kazuko Yorita, Toshiyuki Watanabe, Hideo Misaki, Vincent Massey

The substrate specificity of L-mandelate dehydrogenase fromRhodotorula graminis 483Douglas J. Robertson, Stephen K. Chapman, Graeme A. Reid

(S)-mandelate dehydrogenase from Pseudomonas putida: The roleof the membrane-binding segment and Glycine 81 in the reactivitytoward oxygen 487Yang Xu, Bharati Mitra

3. Acyl-CoA Dehydrogenases

Evolution of an active site:The three-dimensional structures of Acyl-CoA dehydrogenases 491J.-J.P. Kim, M. Wang, R. Paschke, D. Roberts

Bioactivation of 5,6-dichloro-4-thia-5-hexenoyl-CoA by the medium chainAcyl-CoA dehydrogenase:Mechanism-based inactivation by a cytotoxic thioester 499J.F. Baker-Malcolm, C. Thorpe, M.W. Anders, M. Wang, J.-J.P. Kim

Synthesis, activity, and complexation effects of glutaryl-CoA analogswith glutaryl-CoA dehydrogenase 503Emily C. Ferguson, Lisa M. Sharpe, Shannon L. McKinney, Carrie L. Donley,Gregory K. Sewall, Amy L. Larsen, Colleen M. Byron

Redox potential and steady-state kinetic measurements of short-chainAcyl-CoA dehydrogenase (SCAD) active site mutants 507J.D. Pellett, A.K. Saenger, J.A. Fuchs, M.T. Stankovich,D.F. Becker

XXVII

Interactions of rat acyl-CoA oxidase with substrate analogsused as active-site probes 511Haruhiko Tamaoki, Chiaki Setoyama, Kyosuke Sato, Yasuzo Nishina,S. Tanase, Kiyoshi Shiga, Retsu Miura

Biochemical characteristics of recombinant human isovaleryl-CoAdehydrogenase pre-treated with ethylenediaminetetraacetate 515Al-Walid, A. Mohsen, Jerry Vockley

Probing the mechanism of medium-chain acyl-CoA dehydrogenase(MCAD) using spectrally active alternative-substrates and products 519Teresa R. Lamm, Marian Stankovich

Substrate chain length specificity of acyl-CoA dehydrogenases:studies on different mutants 523Burkhard Kiichler, Andy Nandy, Abdel Ghany and Sandro Ghisla

Probing the active site of the medium chain Ayl-CoA dehydrogenase:4-OH-cinnamoyl-CoA as a sensitive probe of polarization and ionization 527Irina Rudik, Colin Thorpe, Alasdair Bell, Peter Tonge

Substrate polarization of medium chain Acyl-CoA dehydrogenase(MCAD) • 531Avery W. Stephens, Kim M. Sabaj, Marian T. Stankovich,A. Bell, Peter J. Tonge

Medium chain Acyl-CoA dehydrogenase genetic defects:identification and partial characterisation of two new patient mutants 535A.G. Abdel Ghany, B. Kuchler, P. Bross, S. Ghisla

The crystal structur of human glutaryl-CoA dehydrogenase 539J.-J.P. Kim, M. Wang, R. Paschke

4. Amino Acid Oxidases

The reaction scheme of D-amino acid oxidase: substrate/productalignment for optimization of reductive and oxidative half-reactions 543Retsu Miura, Chiaki Setoyama, Yasuzo Nishima, Kiyoshi Shiga,Hisashi Mitzutani, Ikuko Miyahara, Ken Hirotsu

Reaction mechanism of flavin dehydrogenation by D-amino acid oxidase 551Loredano Pollegioni, Sandro Ghisla, Stephan Umhau, Gianluca Molla,Christopher M. Harris, Mirella S. Pilone

XXVIII

Structure and function of Rhodotorula gracitts D-amino acid oxidase1. Site-directed mutagenesis of tyrosines 223 and 238 559Gianluca Molla, Christopher M. Harris, Angelo Boselli, Silvia Sacchi,Mirella S. Pilone, Loredano Pollegioni

Structure and function of Rhodotorula gracilis D-amino acid oxidase2. Site-directed mutagenesis of arginine 285 and pH effects 563Viyiana Job, Christopher M. Harris, Davide Porrini, Gianluca Molla,Maria Cristina Vegezzi, Laura Motteran, Sandro Ghisla, Loredano Pollegioni,Mirella S. Pilone

Very high resolution crystal structure of D-amino acid oxidase.Insights into the reaction mechanism and mode of ligand binding 567S. Umhau, K. Diederichs, W. Welte, S. Ghisla, L. Pollegioni, G. Molla,D. Porrini, M.S. Pilone

Mutation in an hydrophobic sequence motif commonto N-hydroxylating enzymes 571Oliver Seth, Liliana Smau, Wolfram Welte, Sandro Ghisla, Peter Macheroux

Oligomerization and aggregation of lysine-N*-hydroxylase -an enzyme of the bacterial aerobactin biosynthesis 575Liliana Smau, Ohver Seth, Wolfram Welte, Sandro Ghisla,Peter Macheroux, Richard Thomas

Gender dependent tissue distribution of D-aspartate oxidasein Xenopus laevis 579Gabriella Tedeschi, Armando Negri, Emanuella Oungre, F. Cecilliani,Severino Ronchi, Giovanni Bernardini

Studies on the glycosylation of L-amino acid oxidase from the Malayanpit viper Calloselasma rhodostoma 583Peter Macheroux, Karina Kitzing, Michael Vetsch, Margarethe Sappelt,Sandro Ghisla, Margarete Schwarz, Manfred Kurfiirst

Role of the hydrogen-bonding network associated with isoalloxazinein the catalysis of D-amino acid oxidase 587Chiaki Setoyama, Retsu Miura, Yasuzo Nishina, Kiyoshi Shiga,Hisashi Mizutani, Ikuko Miyahara, Ken Hirotsu

Comparison of the amino acid sequence of Calloselasma rhodostomaL-amino acid oxidase to other FAD-dependent oxidases 591Oliver Seth, Lo-Chun Au, Sandro Ghisla, Claus Bollschweiler,Manfred Kurfiirst, Peter Macheroux

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Regulation of D-amino acid oxidase expression in the obligatoryaerobic yeast Rhodotorula gracilis 595Simona Rizzi, Gianluca Molla, Sonia Fantinato, Loredano Pollegioni

5. Dihydroorotate Dehydrogenases

Roles of three prosthetic groups in the tetrameric dihydroorotatedehydrogenase B from Lactococcus lactis 599Kaj Frank Jensen, Olof Bjornberg, Finn S. Nielsen, Mette Ottosen,Palle G. S0rensen, Paul Rowland, Sofie N0rager, Sine Larsen

Structure - function relationship of dihydroorotate dehydrogenases 603Sofie N0rager, Sine Larsen, Olof Bjornberg, Kaj Frank Jensen

The dihydroorotate dehydrogenases of Escherichia coli andLactococcus lactis represent two distinct families of the enzyme 607Olof Bjornberg, Kaj Frank Jensen, Anne Charlotte Griiner, Mette Ottosen,Palle Gravegaard S0rensen, Paul Rowland, Sofie N0rager, Sine Larsen

A sticky hydrogen atom in the reaction catalyzed by dihydroorotatedehydrogenase from Saccharomyces cerevisiae 611Douglas B. Jordan, John J. Bisaha, Michael A. Picollelli

Reduction reactions of two dihydroorotate dehydrogenases 615Bruce A. Palfey, Olof Bjornberg, Kaj Frank Jensen

Dihydroorotate dehydrogenase from the thermoacidophilic archaeonSulfolobus solfataricus is a cytosolic dimer 619Palle Gravegaard S0rensen, Gert Dandanell

Reversible unfolding and stability of dihydroorotate dehydrogenase Afrom Lactococcus lactis 623Mette Brimheim Ottosen, Olof Bjdrnberg, Kaj Frank Jensen,Lise Schack, Sine Larsen

6. Various Aspects

Cholesterol oxidase from Brevibacterium sterolicum and Streptomyceshygroscopicus: a covalent FAD binding vs. a non-covalent one 627Laura Motteran, Mirella S. Pilone, Loredano Pollegioni, Sandro Ghisla

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Involvement of Aspl70 in catalysis of vanillyl-alcohol oxidase 631Robert H. H. van den Heuvel, Willem J. H. van Berkel,Marco W. Fraaije, Andrea Mattevi

Dynamics and spatial peculiarities of the mitochondria!NADH dehydrogenase. Spectroscopic studies. 635Nikolai Vekshin, Irina Sharova, Vladislav Sukharev

An iso-mechanism for nitroalkane oxidase: evidence for a slow protontransfer to solvent coupled to isomerization of the free reduced enzyme 639Giovanni Gadda, Paul F. Fitzpatrick

IX. Old Yellow Enzyme

New things about Old Yellow Enzyme 645Vincent Massey, Younus Mean, Dong Xu, Bette Jo Brown

Overexpression and characterization of 12-oxophytodienoic acidreductase from tomato; a member of the OYE family 655Jochen Strassner, Andreas Fiirholz, Peter Macheroux,Nikola Amrhein, A. Schaller, F. Schaller, E.W. Weiler

Study of the function of Old Yellow Enzyme in Saccharomyces cerevisiae 659Bette Jo Brown, Vincent Massey

The Old Yellow Enzyme family of flavoenzymes -comparison of substrate specificity and activity against explosives 663Richard E. Williams, Deborah Rathbone, Neil C. Bruce, Nigel S. Scrutton,Peter C.E. Moody, Stephen Nicklin

Structure and mechanism of an opiate-transforming redox enzyme:morphinone reductase 667Peter C.E. Moody, Daniel H. Craig, Nigel S. Scrutton, A.W. Munro,S.K. Chapman, Neil C. Bruce

Structure and mechanism of an explosive degrading enzyme:pentaerythritol tetranitrate reductase 671T. Barna, P.C.E. Moody, D.H. Craig, N.S. Scrutton, N.C. Bruce

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X. Flavoproteins and -SS-/-SH

An NADH oxidase functional as alkyl hydroperoxide reductase 677Youichi Niimura, Yoshitaka Nishiyama, Kouji Takeda, Hirokazu Tsuji,Kenji Ohnishi, Toshihiro Watanabe, Takeshi Nishino, Vincent Massey

Functional characterization of the N-terminus of AhpFby chimeric construction with TrR 681C. Michael Reynold, Leslie B. Poole

Egg white sulfhydryl oxidase: convergent evolution and catalysis ofdisulfide bond formation in proteins and peptides 685Karen L. Hoober, Colin Thorpe, D.L. Coppock

Functional and regulatory studies of two distinct NADH oxidasesfrom Streptococcus mutans 691M. Higuchi, Y. Yamamoto, L. Poole, M. Shimada, Y. Sato,N. Takahashi, Y. Kamio

Thioredoxin reductase from Plasmodiumfalciparum:interaction between the C-terminal cysteine residues and theactive site disulfide/dithiol 695Pan-Fen Wang, L. David Arscott, Charles H. Williams, Jr . 'Tim-Wolf Gilberger, Sylke Muller

Characterization of a glutathion amide reductase from the purplephototrophic bacterium Chromatium gracile, its altered substratespecificity, and involvement in detoxification of oxygen 699B. Vergauwen, Y. Guisez, R.G. Bartsch, T.E. Meyer, M.A. Cusanovich,J.J. van Beeumen

Tetryl as inhibitor and "subversive substrate" for human erythrocyteglutathione reductase 703V. Miskiniene, Z. Anusevicius, A. Maroziene, R. Kliukiene, H. Nivinskas,J. Sarlauskas, N. Cenas, K. Becker

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XI. Protein-Protein Interactions

Reaction and substrate recognition of the flavin reductase FRase Ifrom bioluminescent bacterium Vibrio fischeri:biochemistry and X-ray crystallography of FRase I 709W.C. Lee, T. Hirai, T.Kobori, H.Sasaki, H. Koike, M. Tanokura, S. Zenno,K. Saigo, T. Nishino, M.E.P. Murphy, E.T. Adman

The application of QM/MM techniques to flavoproteins:p-hydroxybenzoate hydroxylase (PHBH) 719Lars Ridder, Jacques Vervoort, Ivonne M. C. M. Rietjens

Structure and function of adrenodoxin reductase 729Gabriele A. Ziegler, Clemens Vonrhein, Georg E. Schulz, Israel Hanukoglu

Properties of NifL, a regulatory flavoprotein containing a PAS-domain 737R. Little, S. Hill, S. Perry, S. Austin, F. Reyes-Ramirez,R. Dixon, P. Macheroux

Molecular recognition between ferredoxin- NADP* reductase and itsprotein partners: role of charged and hydrophobic residues 741M. Martinez Jiilvez, I. Nogues, M. Medina, C. G6mez-Moreno,J.K. Hurly, G. Tollin

Analysis of a trimeric complex involving chorismate synthasefrom Bacillus subtilis 749Teresa Fitzpatrick, Nikolaus Amrhein, Peter Macheroux

Regulation of electron transfer in flavocytochrome b588 by theactivation domain of p61phox 753Yukio Nisimoto, Chang-Hoon Han, Shabnam Motalebi, David J. Lambeth

XII. Multidomain Flavoproteins

A new type of flavin adenine dinucleotide-binding resolved in themolybdo iron-sulfur-flavoprotein carbon monoxide dehydrogenasefrom Oligotropha carboxidovorans 759L. Gremer, Sabine Kellner, Ortwin Meyer,Holger Dobbek, Robert Huber

XXXIII

Studies on the iron-sulfur flavoenzyme adenosine 5'-phosphosulfatereductase from different organisms reveals a common structure 767and mechanismGunter Fritz, Thomas Buchert, Peter M. H. Kroneck

Properties of xanthine oxidase from human milk: the enzyme isgrossly deficient in molybdenum and substantially deficientin iron-sulphur centres 775Robert C. Bray, David Lowe, Ben Godber, Roger Harrison, Robert Eisenthal

Respiratory complex II from the thermoacidophilic archaeon,Sulfolobus sp. strain 7: genes and protein 779Toshio Iwasaki, Miho Aoshima, Asako Kounosu, Tairo Oshima

Crystal structur of xanthine oxidoreductase andERP assignment of Fe/S centers 783Cristofer Enroth, Bryan T. Eger, Emil F. Pai, Ken Okamoto, Toshio Iwasaki,Tomoko Nishino, Hiroyuki Hori, Takeshi Nishino

Azospirillum brasilense glutamate synthase: identification of substratesand cofactors binding sites by site-directed mutagenesis 787P. Morandi, B. Valzasina, C. Colombo, M.A. Vanoni, B. Curti

The mechanism of conversion from xanthine dehydrogenase to oxidaseof rat enzyme studied by site-directed mutagenesis 791Tomoko Nishino, Ken Okamoto, Hiroyuki Hori,Asako Kounosu, Takeshi Nishino

Studies of the bile acid inducible NADH: flavin oxidoreductase 795Martin Mewies, RussJClle

Azospirillum brasilense glutamate synthase: overproduction inE. coli and characterization of the recombinant enzyme 799M.A. Vanoni, S. Ravasio, H. Stabile, R. Bossi, G. Zanetti, B. Curti

Towards the natural electron donor of adenosine 5'-phosphosulfate (APS)reductase from Desulfovibrio desulfuricans Essex 803Thomas Buchert, Gunter Fritz, Peter M. H. Kroneck

xxxrv

XIII. Biosynthetic and Metabolic Aspects

19F NMR studies on lumazine synthase from Bacillus subtilis 809Markus Fischer, Karl Kugelbrey, Johannes Scheming, Mark Cushman

Reconstitution of 6-hydroxy-D-nicotine oxidase holoenzyme withN6-[N-(2-methylferrocene)-caproylamidoethyl]-FAD 813Roderich Brandsch, Carmen Brizio, Andreas F. Biickmann

Riboflavin transport and metabolism in isolated mitochondria 817Maria Barile, Carmen Brizio, Caterina De Virgilio, Salvatore Passarella

Synthesis and application of FAD analogs, functionalized atthe Imposition of the adenine 821A. Stocker, H.-J. Hecht, A.F. Buckmann, E. Katz, I. Willner,M. Dosch, C. Troupe

Biosynthesis of riboflavin: NMR studies on the 46-kDa dimeric protein,3,4-dihydroxy-2-butanone 4-phosphate synthase 825G. Richter, C. Krieger, M. Kelly, Y. Yu, L.J. Ball, P. Schmieder, H. Oschkinat

Investigation of the binding of 6,7-bis(trifluoromethyl)-8-ribityllumazinesto 15N-labeled lumazine synthase by REDOR-NMR 829Markus Fischer, Jon M. Goetz, Barbara Poliks, Daniel R. Studelska,Andreas GieBauf, Karl Kugelbrey, Mark Cushman, Jakob Schaefer

Biosynthesis of riboflavin: the reaction catalysed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without catalysis underphysiological conditions. 833Klaus Kis, KarlKugelbrey, Adelbert Bacher,

XIV. Medical Aspects

Medical aspects of flavins and flavoproteins - An outlook 839Kunio Yagi

Structural aspects of the flavoprotein domains of isoformsof nitric oxide synthase 845B.S. Masters, R.T. Miller, P. Martasek, L.J. Roman, J.S. Nishimura,S. Panda, D.E. Harris, P.M. Horowitz, T.M. Shea, J.C. Salerno,J. Zhang, J.J.P. Kim

XXXV

Family traits of FAD-containing disulfide reductases as drug targets 853K. Becker, S. Kanzok, R. Iozef, I. Tiirbachova, R.H. Schirmer

Disulfide reductases are destabilized by physiologic concentrationsofNADPH 857M. Schirmer, M. Scheiwein, S. Gromer, K. Becker, R.H: Schirmer

Construction of separate expression vectors for the catalytic core andthe N-terminal metal binding domain of Tn501 mercuric ion reductase 863Susan M. Miller

D-amino acid oxidase activity in the senescence-accelerated mouse 871Nobuko Ohishi, Shin-ichiro Yokoyama, Miyuki Kurata,Masashi Tanaka, Sadaaki Komura, Kunio Yagi

Riboflavin derivatives in food 875Anna Gliszczynska, Anna Koziolowa

A covalently bound flavin discovered in the reductase componentof a tetrahydrofuran-induced multicomponent monooxygenase 879Barbara Thiemer, Jan R. Andreesen, Thomas Schrader

Study on vanillyl-alcohol oxidase reveals a novel (covalent)flavoprotein family 883Marco W. Fraaije, Robert H.H. van den Heuvel,Willem J.H. van Berkel, Andrea Mattevi

Gene expression of D-amino acid oxidase in nervous system 887T. Kanamori, M. Obayashi, O. Jinnouchi, K. Kanda, Y. Urai,Y. Shishido, A. Suzue,.T. Sakai, K Fukui

Characterization of an adrenodoxin reductase-like protein ofMycobacterium tuberculosis 891A. Aliverti, F. Fischer, S. Pasquini, M.A. Vanoni, B. Curti, G. ZanettiR. Cantoni, M. Branzoni.G. Riccardi

Irreversible inhibitors of T. cruzi trypanothione reductase:Kinetic and crystallographic studies 895Susanne Bonse, R. Louise Krauth-Siegel, lime Schlichting, Gordon Lowe

Glutathione reductase of Plasmodiumfalciparum -Reductive and oxidative half reactions 899C.C. Bohme, R.H. Schirmer, K. Becker, L.D. Arscott, C.H. Williams Jr.

XXXVI

Cloning and mapping of the cDNA for human sarcosine dehydrogenase;a flavoenzyme defective in patients with sarcosinemia 903M. Eschenbrenner, M.S. Jorns

Author Index 909

Subject Index 917

Participants 931