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FLAVINS AND 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 PUBLICATIONS BERLIN 1999 XV Contents

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

Living through various phases of flavin research 3 Helmut Beinert

Synthetic Models of flavoenzyme activity 17 Vincent Rotello NMR-studies of flavocytochrome b2 reconstituted with 15N, I3C labelled flavin Garit Fleischmann, Franz Miiller, Heinz Riiterjans, Florence Lederer

One and two electron redox cycles in flavin-dependent dehydrations 31 W. 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 using Stark-effect spectroscopy ' 41 Robert J. Stanley, Haishan Jang

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

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

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

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

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

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

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

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

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

II. Spectroscopy

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

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

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

Direct measurements of ultrafast excited state quenching of isoalloxazine by adenine in FAD 99 Robert J. Stanley, Alexander W. MacFarlane IV XVII III. Flavins and Electrons

1. Electron-Transfer in Heme-containing Systems

Flavocytochrome c3: The structure and mechanism revealed 105 S.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 thiosulfate utilization gene cluster from the green phototrophic bacterium, Chlorobium limicola (strain Tassajara) 115 Fabienne Verte, Yves Guisez, Terrance E. Meyer, Michael A.Cusanovich Jozef J. Van Beeumen

A flavohemoprotein from the cellulolytic fungus Humicola insolvens contains 6-hydroxy-FAD as the dominant active 119 Kiyohiko 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 123 Christopher G. Mowat, Stephen K. Chapman, Caroline S. Miles, Graeme A. Reid, A.W. Munro

Cloning and characterization of a membrane-bound flavocytochrome c from the purple phototrophic sulfur bacterium Ectothiorhodospira vacuolata 127 Vesna 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 131 Jeffrey A. Mertens, Wilbur H. Campbell, Lawrie Skipper, David J. Lowe

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

The base of the fumarate reductase from Shewanellaftigidimarina 139 Mary K. Doherty, Stephen K. Chapman Caroline S. Miles, Graeme A. Reid XVIII

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

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

The flavoproteindomain s of nitric oxide synthase isoforms: site of possible regulatory control of flavin to heme electron transfer 151 R. 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 NADPH- cytochrome P450 reductases and its domains 155 Andrew W. Munro, T. McSorley, Michael A. Noble, Laura Robledo, Stephen K. Chapman

Crystallographic insights into the hydride transfer mechanism of NADPH-cytochrome P450 159 Paul A. Hubbard, Rosemary Paschke, Jung-Ja P. Kim Anna L. Shen, Charles B. Kasper

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

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

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

2. Electron Transfer in Flavodoxins

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

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

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

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

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

3. Various Aspects

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

Overexpression of the bifunctional chorismate sythase of Neurospora crassa 203 Karina 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 207 Shigenobu Kimura, Masanori Kawamura, Takashi Iyanagi

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

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

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

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

Evidence that Megasphaera elsdenii synthesises two different electron-transferring flavoproteins 227 Zuhair Nasrallah, Hugh O'Neill, Stephen G. Mayhew IV. 3D-Structures of Flavoproteins

Structural flavinology on the brink 233 P. Andrew Karplus

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

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

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

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

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

A new functional model for the Escherichia coli sulfite reductase: the aipi complex 259 Jaques Coves, Mahel Zeghouf, Marc Fontecave XXI V. Flavins and Pyridine Nucleotides

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

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

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

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

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

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

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

Determinants of cofactor specificity in Anabaena PCC7119 ferredoxin-NADP+-reductase 293 Jesus Tejero, Alejandra Luquita, Koert Grever, Carlos G6mez-Moreno, Milagros Medina, R. Perham XXII VI. Flavins and Light

1. Photochemistry, Blue Light Effects and Photolyases

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

DNA photolyase and cryptochrome 309 Takeshi Todo

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

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

2.

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

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

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

VII. Flavins and

1. General Aspects

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

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

2. Monooxygenases

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

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

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

Purification and some properties of acetophenone monooxygenase 375 Marielle 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 379 Mariliz Ortiz-Maldonado, Sara Aeschliman, David P. Ballou, Vincent Massey

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

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

3. Various Aspects

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

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

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

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

What protein features makep-hydroxybenzoate hydroxylase react rapidly with oxygen? 409 Willem 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 of monomeric 415 M.S. Jorns, M.A. Wagner, P. Trickey, F.S. Mathews

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

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

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

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

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

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

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

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

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

2. a-Hydroxy Acid Dehydrogenases

X-ray studies of recombinant rat kidney long-chain hydroxy acid oxidase and of the recombinant flavin-binding domain of bakers yeast flavocytochrome b2 459 L.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 of p-halogeno a-hydroxy acids catalysed by FMN-dependent hydroxy acid-oxidizing enzymes: a mutational analysis with flavocytochrome b2 463 Sabrina Bodevin, Florence Lederer

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

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

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

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

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

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

3. Acyl-CoA Dehydrogenases

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

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

Synthesis, activity, and complexation effects of glutaryl-CoA analogs with glutaryl-CoA dehydrogenase 503 Emily 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-chain Acyl-CoA dehydrogenase (SCAD) active site mutants 507 J.D. Pellett, A.K. Saenger, J.A. Fuchs, M.T. Stankovich,D.F. Becker XXVII

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

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

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

Substrate chain length specificity of acyl-CoA dehydrogenases: studies on different mutants 523 Burkhard 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 527 Irina Rudik, Colin Thorpe, Alasdair Bell, Peter Tonge

Substrate polarization of medium chain Acyl-CoA dehydrogenase (MCAD) • 531 Avery 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 535 A.G. Abdel Ghany, B. Kuchler, P. Bross, S. Ghisla

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

4. Amino Acid Oxidases

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

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

Structure and function of Rhodotorula gracitts D-amino acid oxidase 1. Site-directed mutagenesis of tyrosines 223 and 238 559 Gianluca Molla, Christopher M. Harris, Angelo Boselli, Silvia Sacchi, Mirella S. Pilone, Loredano Pollegioni

Structure and function of Rhodotorula gracilis D-amino acid oxidase 2. Site-directed mutagenesis of arginine 285 and pH effects 563 Viyiana 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 567 S. Umhau, K. Diederichs, W. Welte, S. Ghisla, L. Pollegioni, G. Molla, D. Porrini, M.S. Pilone

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

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

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

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

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

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

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

5. Dihydroorotate Dehydrogenases

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

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

The dihydroorotate dehydrogenases of Escherichia coli and Lactococcus lactis represent two distinct families of the enzyme 607 Olof 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 dihydroorotate dehydrogenase from Saccharomyces cerevisiae 611 Douglas B. Jordan, John J. Bisaha, Michael A. Picollelli

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

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

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

6. Various Aspects

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

Involvement of Aspl70 in catalysis of vanillyl-alcohol oxidase 631 Robert 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. 635 Nikolai Vekshin, Irina Sharova, Vladislav Sukharev

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

IX. Old Yellow Enzyme

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

Overexpression and characterization of 12-oxophytodienoic acid reductase from tomato; a member of the OYE family 655 Jochen 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 659 Bette Jo Brown, Vincent Massey

The Old Yellow Enzyme family of flavoenzymes - comparison of substrate specificity and activity against explosives 663 Richard 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 667 Peter 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 671 T. Barna, P.C.E. Moody, D.H. Craig, N.S. Scrutton, N.C. Bruce XXXI

X. Flavoproteins and -SS-/-SH

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

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

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

Functional and regulatory studies of two distinct NADH oxidases from Streptococcus mutans 691 M. 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 the active site disulfide/dithiol 695 Pan-Fen Wang, L. David Arscott, Charles H. Williams, Jr.' Tim-Wolf Gilberger, Sylke Muller

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

Tetryl as inhibitor and "subversive substrate" for human erythrocyte glutathione reductase 703 V. Miskiniene, Z. Anusevicius, A. Maroziene, R. Kliukiene, H. Nivinskas, J. Sarlauskas, N. Cenas, K. Becker XXXII XI. Protein-Protein Interactions

Reaction and substrate recognition of the flavin reductase FRase I from bioluminescent bacterium Vibrio fischeri: biochemistry and X-ray crystallography of FRase I 709 W.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) 719 Lars Ridder, Jacques Vervoort, Ivonne M. C. M. Rietjens

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

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

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

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

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

XII. Multidomain Flavoproteins

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

Studies on the iron-sulfur flavoenzyme adenosine 5'-phosphosulfate reductase from different reveals a common structure 767 and mechanism Gunter Fritz, Thomas Buchert, Peter M. H. Kroneck

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

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

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

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

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

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

Azospirillum brasilense glutamate synthase: overproduction in E. coli and characterization of the recombinant enzyme 799 M.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 803 Thomas Buchert, Gunter Fritz, Peter M. H. Kroneck xxxrv XIII. Biosynthetic and Metabolic Aspects

19F NMR studies on lumazine synthase from Bacillus subtilis 809 Markus Fischer, Karl Kugelbrey, Johannes Scheming, Mark Cushman Reconstitution of 6-hydroxy-D-nicotine oxidase holoenzyme with N6-[N-(2-methylferrocene)-caproylamidoethyl]-FAD 813 Roderich Brandsch, Carmen Brizio, Andreas F. Biickmann

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

Synthesis and application of FAD analogs, functionalized at the Imposition of the adenine 821 A. 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 825 G. Richter, C. Krieger, M. Kelly, Y. Yu, L.J. Ball, P. Schmieder, H. Oschkinat

Investigation of the binding of 6,7-bis(trifluoromethyl)-8-ribityllumazines to 15N-labeled lumazine synthase by REDOR-NMR 829 Markus 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 under physiological conditions. 833 Klaus Kis, KarlKugelbrey, Adelbert Bacher, XIV. Medical Aspects

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

Structural aspects of the flavoprotein domains of isoforms of nitric oxide synthase 845 B.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 853 K. Becker, S. Kanzok, R. Iozef, I. Tiirbachova, R.H. Schirmer

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

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

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

Riboflavin derivatives in food 875 Anna Gliszczynska, Anna Koziolowa

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

Study on vanillyl-alcohol oxidase reveals a novel (covalent) flavoprotein family 883 Marco 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 887 T. Kanamori, M. Obayashi, O. Jinnouchi, K. Kanda, Y. Urai, Y. Shishido, A. Suzue,.T. Sakai, K Fukui

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

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

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

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

Author Index 909

Subject Index 917

Participants 931