Flavins and Flavoproteins Flavins and Flavoproteins Proceedings of the Eighth International Symposium Brighton, England, July 9 -13,1984 Editors R. C. Bray • P C. Engel • S. G. Mayhew W DE G Walter de Gruyter • Berlin • New York 1984 Editors Robert C. Bray, B. A.,Ph. D., Sc. D. The University of Sussex School of Molecular Sciences Brighton BN1 9QJ Great Britain Paul C.Engel, M. A.,D.Phil. Department of Biochemistry University of Sheffield Sheffield S10 2TN Great Britain Stephen G. Mayhew, B. Sc.,Ph. D. Department of Biochemistry University College Dublin Belfield, Dublin 4 Ireland Library of Congress Cataloging in Publication Data Flavins and flavoproteins. Includes bibliographies and indexes. 1. Flavins-Congresses. 2. Flavoproteins-Congresses. I. Bray, R. C. (Robert C.), 1928- . II. Engel, Paul C. III. Mayhew, S. G. (Stephen G.), 1940- . IV International Symposium on Flavins and Flavoproteins (8th: 1984: Brighton, East Sussex) QP67I.F5F53 1984 574.19'218 84-23034 ISBN 3-11-009879-2 CIP-Kurztitelaufnahme der Deutschen Bibliothek Flavins and flavoproteins : proceedings of the internat, symposium. - Berlin ; New York : de Gruyter 8. Brighton, England, July 9-13.1984. - 1984. ISBN 3-11-009879-2 Copyright © 1984 by Walter de Gruyter& Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Dieter Mikolai, Berlin. - Printed in Germany. PREFACE The Eighth International Symposium on Flavins and Flavo- proteins was held at the University of Sussex (Brighton, England) from the 8-13 July 1984. This coincided with an important anniversary for the flavin world, the 50th anniversary of the synthesis of lumiflavin by Richard Kuhn in 1934 . The organisers are indebted to a small but select group of bodies listed elsewhere who contributed financially to the anniversary symposium. In particular substantial help (though at an alarmingly late date) from the International Union of Biochemistry (through the Interest Group on Oxi- dases and Redox Enzymes) made it possible to provide travel support, if on a somewhat nominal scale, to all speakers who required it and to a few others. Support from other sources made it possible to subsidise registration fees, particularly for an encouragingly large contingent of younger participants; it is in their hands that the future development of flavin research will rest. It has to be said that many firms, esp- cially in the pharmaceutical industry, to whom we turned for support were unable to help, and this was disappointing. These firms are among the most obvious beneficiaries of research into a group of enzymes that not only includes prime drug targets but also is intimately involved in drug metabolism. In view of the minimal support we were able to offer to senior scientists we had serious doubts about likely attendance until a month or two before the meeting. Under these circumstances it was especially gratifying that, in the event, so many travelled from all over the world to take part in the symposium. In planning the programme the Organising Committee decided to continue the pattern adopted at the Ann Arbor Meeting, of morning lectures, afternoon poster sessions and evening VI discussions. This stamina-taxing regime again proved successful and the evening sessions were well attended throughout. The usefulness of the evening sessions owed much to the efforts of the discussion leaders, who receive our special thanks : Martha Ludwig, Franz Muller, Steve Mayhew, Bruno Curti, Graham Palmer, Dale Edmondson. The rigours of the British licensing laws meant that the dis- cussions tended to finish after "closing time" in the outside world, but the bar extension allowed opportunities for continued discussion well into the night. This was the first time that the 'Flavins Symposium' had been held in Britain. Our famed weather lived up to its reputa- tion and had overseas participants in total confusion by the end of the week. On the whole, however, we were lucky and we were certainly spared the alleged divine anger that led to York Minster being struck by lightning in the same week! It rained while we talked science, but the sun always managed to come out for our various excursions. The Chairman would like to thank his colleagues on the Organising Committee for their help and support. Thanks particularly are due to Vince Massey for his guidance which helped to ensure that the meeting followed faithfully in the traditions of earlier ones of this series. The international character of the Organising Committee made it inevitable that there should also be a Local Committee charged with much of the immediate decision-making. However, in this context, "local" encompassed divers parts of the British Isles. Thus it came about that Barry Smith (who has never worked on flavins) was cajoled into joining the Local Committee, as the representative of the Molecular Enzymology Group of the Biochemical Society, and then into shouldering a substantial part of the organisational work during the meeting. The success of the symposium as a whole was due in no small measure to his efforts and those of his helpers, not to VII mention the generous hospitality of the School of Chemistry and Molecular Sciences. Especial thanks are also due to the long-suffering Conference Secretary, Mrs. Margaret Stovold. Each symposium seems to witness the application of a new crop of techniques to the flavin field. This time the impact of recombinant DNA techniques was in evidence for the first time, with the cloning of a number of flavoproteins being reported and detailed structural comparisons based upon DNA sequencing being presented. The advances in the application of high-resolution n.m.r. techniques were also prominent. The use of flavin analogues with chemically reactive substituent groups formed the subject of numerous communications and has justified a separate section in our division of the contents of this book. The task of sorting the diverse contributions into categories has been extremely difficult since, inevitably, so many articles belong in two or more categories. The editors have had to make some very arbitrary decisions but hope that some logic may be discerned in the groupings at which they have finally arrived. This is the first time that the symposium volume has been produced from camera-ready copy. This has entailed some problems for authors and editors alike. Only time will show whether these were justified by unpreceden- tedly rapid publication. We hope so! One of our intended session chairmen, Henry Kamin, was prevented by illness from attending the Symposium. His thoughtful comments were missed, and we wish him a speedy recovery, The next symposium is to be held at Emory University, Atlanta Georgia in 1987. We look forward to many exciting develop- ments over the intervening three years. The flavoprotein V i l i field is as vigorous as ever, and we feel that the pioneers of 1934 would be pleased to see where their researches have led. R.C. Bray P.C. Engel S.G. Mayhew Brighton Sheffield Dublin August 198 4 ORGANISING COMMITTEE LOCAL COMMITTEE H. Beinert (Madison) B. Curti (Milano) R.C. Bray (Brighton) Chairman P.C. Engel (Sheffield) S.G. Mayhew (Dublin) B.E. Smith* (Brighton) S. Ghisla (Konstanz) J.-M. Lhoste (Orsay) V. Massey (Ann Arbor) * representing the Molecular Enzymology Group of the Biochemical Society. D.B. McCormick (Atlanta) G.E. Schulz (Heidelberg) C. Veeger (Wageningen) C. Walsh (Cambridge, MA) C. H. Williams, Jr. (Ann Arbor) K. Yagi (Nagoya) The organisers gratefully acknowledge the financial support received from the following organisations: International Union of Biochemistry Biochemical Society The Royal Society Roche Products The Wellcome Foundation I.C.I. Pharmaceuticals Division ACKNOWLEDGEMENTS CONTENTS P A R T I C I P A N T S x x x i F L A V I N CHEMISTRY The i n f l u e n c e of h y d r o g e n bond formation with the N ( l ) atom on the o r b i t a l s t r u c t u r e of f l a v i n F. M u l l e r , J.K. Eweg, V. S z c z e s n a and B. Hesper 3 Spectral and photochemical properties of a l l o x a z i n e s J. Kozio-f, A. Kozioiowa, W. B a b s , J. D a w i d o w s k i , D. P a n e k - J a n c , M. S t r o i f i s k a , V. S z c z e s n a , H. S z y m u s i a k and B. T y r a k o w s k a 17 n.. . n - i n t e r a c t i o n s of f l a v i n s : novel coenzyme models o f the c y c l o p h a n e t y p e M . F . Z i p p l i e s and H. A. S t a a b 21 A l a s e r f l a s h p h o t o l y s i s s t u d y of the t r i p l e t states of lumichromes P . F . Heelis and G.O. P h i l l i p s 25 The d a r k formation of r a d i c a l s in f l a v i n i u m c a t i o n / a c i d s y s t e m s R. A d d i n k and H . I . X . Mager 29 ENDOR s t u d i e s on f l a v i n r a d i c a l s M. Bock, M. E i s n e r and H. K u r r e c k 33 On the role of some f l a v i n adducts as one-electron donors H . I . X . Mager and R. A d d i n k 37 XII P h o t o i n a c t i v a t i o n of f l a v i n redox c a t a l y s i s : the r e d u c t i v e f l a v i n photoadduct formation U. Ott, R. Traber and H . E . A . Kramer 41 F l a v i n oxygen chemistry brought to date T . C . Bruice 45 Pulse r a d i o l y s i s studies on the e q u i l i b r i a between reduced and o x i d i z e d free f l a v i n species and the effect of molecular oxygen R . F . Anderson 57 Effect of pH on the o x i d a t i o n - r e d u c t i o n properties of 8a-imidazole f l a vi ns G. Williamson and D.E. Edmondson 61 Studies of intermediates i n reactions of f l a v i n s and s u l p h y d r y l compounds P.S. Surdhar and D.A. Armstrong 67 A kinetic study on the a c i d - c a t a l y s e d phosphate migration i n r i b o f l a v i n phosphates P. Nielsen, P. Rauschenbach and A. Bacher 71 Chemical s t r u c t u r e of n e k o f l a v i n K. Matsui and S. Kasai 75 F L A V O P R O T E I N S T R U C T U R E B i n d i n g mode and action of FAD i n glutathione reductase G.E. Schulz 81 XIII Active site c h e m i c a l m o d i f i c a t i o n and s e q u e n c i n g of f l a v o p r o t e i n s C.H. W i l l i a m s , J r . , L . D . Arscott and R . P . Swenson 95 M o l e c u l a r genetic a p p r o a c h e s to the s t u d y of E. c o l i f l a v o p r o t e i n s J.R. Guest and D.W. Rice I l l G l u t a t h i o n e r e d u c t a s e : mutation, c l o n i n g and sequence a n a l y s i s of the gene i n E. c o l i S. Greer and R.N. Perham 125 The coenzyme b i n d i n g site of g l u t a t h i o n e r e d u c t a s e . C o r r e l a t i o n of X - r a y s t u d i e s with kinetic d a t a E . F . P a i , E. Horn and G.E. S c h u l z 139 13 C-NMR s t u d y on the active sites of l i p o a m i d e d e h y d r o g e n a s e and g l u t a t h i o n e r e d u c t a s e W . A . M . v a n den B e r g , J. V e r v o o r t , C .T .W. Moonen, F. M u l l e r , I . C a r l b e r g and B. M a n n e r v i k 143 X - r a y c r y s t a l l o g r a p h i c s t u d i e s on l i p o a m i d e d e h y d r o g e n a s e from Azotobacter v i n e l a n d i i A.J. S c h i e r b e e k , J. Drenth and W.G.J. Hoi 147 M o b i l i t y of l i p o a m i d e d e h y d r o g e n a s e i n and out of the p y r u v a t e d e h y d r o g e n a s e complex from Azotobacter v i n e l a n d i i A. de Kok and A.J.W.G. V i s s e r 149 P a r t i a l amino acid sequence of p i g heart l i p o a m i d e d e h y d r o g e n a s e L . D . Arscott, M. Berman and C.H. W i l l i a m s , Jr 153 The amino a c i d sequence e n c o m p a s s i n g the active site h i s t i d i n e r e s i d u e of l i p o a m i d e d e h y d r o g e n a s e from Esc heric hi a c o l i l a b e l l e d with a b i f u n c t i o n a l a r s e n o x i d e C . F . B . Holmes and K.J. S t e v e n s o n 157 XIV I n a c t i v a t i o n of p i g h e a r t l i p o a m i d e d e h y d r o g e n a s e by 1 , 3 - di bromoacetone J . S . Lee and C.H. W i l l i a m s Jr 161 The e v o l u t i o n of m e r c u r i c r e d u c t a s e , and a r e d o x t r a n s f e r model for m e r c u r i c i o n d e t o x i f i c a t i o n i n b a c t e r i a N . L . Brown and D. Goddette 165 S t r u c t u r a l s t u d i e s on f e r r e d o x i n - N A D P + o x i d o r e d u c t a s e from S p i r u l i n a , a b l u e - g r e e n a l g a K. W a d a , Y. Vao, T. T a m u r a , H. M a t s u b a r a and K. Kodo 169 The amino a c i d sequence and p a r t i a l t e r t i a r y s t r u c t u r e of f e r r e d o x i n - N A D P + o x i d o r e d u c t a s e from s p i n a c h P . A . K a r p l u s , J . R . H e r r i o t t and K. A. W a l s h 173 On the n a t u r e of f e r r e d o x i n : f e r r e d o x i n - N A D P + r e d u c t a s e c o m p l e x M. S h i n a n d N. S a k i h a m a 175 P r o p e r t i e s of a c r o s s - l i n k e d complex between f e r r e d o x i n - N A D P + r e d u c t a s e and f e r r e d o x i n G. Zanetti and B. C u r t i 179 On the e n i g m a of o l d yellow e n z y m e ' s s p e c t r a l p r o p e r t i e s J . K . Eweg, F. M u l l e r , W.H. v a n Berkel and B. Hesper 183 "On the e n i g m a of o l d yellow e n z y m e ' s s p e c t r a l p r o p e r t i e s " V. M a s s e y , L . M . S c h o p f e r and W.R. Dunham 191 NMR s t u d i e s on the o l d yellow enzyme W-D B e i n e r t , H. R u t e r j a n s and F. M u l l e r 211 XV Structural and kinetic c h a r a c t e r i s t i c s of dimethyl glycine dehydrogenase and s a r c o s i n e dehydrogenase R.J. Cook, D.H. Porter, K.S. Misono and C. Wagner 215 Evidence for two s p a t i a l l y distinct domains on each s u b u n i t of methylenetetrahydrofolate reductase R.G. Matthews, M. Vanoni, S. Khani, J.F. Hainfeld and J. Wall. 217 Structure of NADH-cytochrome b^ reductase of human erythrocytes T. Y u b i s u i , T. M i y a t a , S. I w a n a g a , M. Tamura, S. Voshida and M. T a k e s h i t a 221 Structural comparison of the s u c c i n a t e dehydrogenase and fumarate reductase of E s c h e r i c h i a coli J.R. Guest, M. G. D a r l i s o n , R.J. Wilde and D. Wood 225 Lack of assembly of succinate and NADH-ubiquinone oxidoreductases in i r o n - d e f i c i e n t r a t skeletal muscle mitochondria B.A.C. A c k r e l l , B. Cochran, K. L a r s o n , E.B. Kearney, J. M a g u i r e and P.R. Dallman 229 C r y s t a l structure s t u d y of trimethylamine dehydrogenase F.S. Mathews, L.W. Lim and N. Shamala 233 The f l a v i n domain of a s s i m i l a t o r y NADH: nitrate reductase from Chlorella v u l g a r i s L.P. Solomonson, and M.J. Barber 247 Polarized absorption spectra of flavocytochrome b 2 s i n g l e c r y s t a l s A. Mozzarelli, M. Tegoni and G.L. Rossi 251 FMN-protein interactions in f l a v o d o x i n from A. n i d u l a n s M.L. Ludwig, K. A. Pattridge and G. Tarr 253 XVI Photochemical f o r m a t i o n of a s t a b l e r e d d e r i v a t i v e of f l a v o d o x i n S . G . Mayhew and V. M a s s e y 261 13 15 D e s u l f o v i b r i o v u l g a r i s f l a v o d o x i n . A C and N NMR i n v e s t i g a t i o n J. V e r v o o r t , F. M ü l l e r , J. L e G a l l , A. B a c h e r and H. S e d l m a i e r . 269 1 3 C-NMR s t u d y on the i n t e r a c t i o n of r i b o f l a v i n with r i b o f l a v i n - b i n d i n g protein R. M i u r a , H. Tojo, S. F u j i i , T. Yamano and Y. M i y a k e 273 The s t r u c t u r e of g l y c o l a t e o x i d a s e from s p i n a c h Y. L i n d q v i s t and C - I . B r ä n d e n 277 ENDOR s t u d i e s of f l a v o p r o t e i n s N. Bretz and H. K u r r e c k 289 Resonance Raman s t u d y on the c o m p l e x e s of D - a m i n o a c i d o x i d a s e Y. N i s h i n a , K. S h i g a , R. M i u r a , H. Tojo, Y. M i y a k e , T. Yamano and H. W a t a r i 291 The role of a r g i n i n e s i n D - a m i n o a c i d o x i d a s e M . P . S i m o n e t t a , S, G a l l i a n i , M . A. V a n o n i , S. Ronchi and B. C u r t i 295 The effect of the m e t h y l a t i o n of h i s t i d i n e - 2 1 7 i n p i g k i d n e y D - a m i n o a c i d o x i d a s e on l i g a n d b i n d i n g and on c a t a l y s i s R . P . S w e n s o n , C.H. W i l l i a m s , Jr. and V. M a s s e y 301 S t o i c h i o m e t r y of the s e l f - a s s o c i a t i o n of D - a m i n o acid o x i d a s e H. Tojo, K. H o r i i k e , K. S h i g a , Y. N i s h i n a , H. W a t a r i and T. Yamano 305 XVII 31 P NMR a n d c h e m i c a l s t u d i e s o n t h e p h o s p h o r u s r e s i d u e s i n m i l k x a n t h i n e o x i d a s e D . E . E d m o n d s o n , M . D . D a v i s a n d F. M ü l l e r 309 S p e c i f i c m o d i f i c a t i o n o f N A D + b i n d i n g s i t e o f c h i c k e n l i v e r x a n t h i n e o x i d a s e w i t h 5 ' - p - f l u o r o - s u l p h o n y l b e n z o y l a d e n o s i n e T. N i s h i n o , T. N i s h i n o a n d K. T s u s h i m a 319 S t u d i e s by e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s p e c t r o s c o p y o f t h e e n v i r o n m e n t o f t h e metal i n t h e m o l y b d e n u m c o f a c t o r from x a n t h i n e o x i d a s e T . R . H a w k e s a n d R . C . B r a y 323 C o u p l i n g between M o ( V ) a n d r e d u c e d Fe/S 1 c e n t r e s i n a l d e h y d e o x i d a s e a n d x a n t h i n e o x i d a s e G . N . G e o r g e 325 P a r t i a l a m i n o a c i d s e q u e n c e o f L - l a c t a t e o x i d a s e from M. smegm a t i s D. A. G i e g e l , V. M a s s e y a n d C . H . W i l l i a m s , Jr 331 C h e m i c a l m o d i f i c a t i o n o f s u l p h y d r y l g r o u p s i n p - h y d r o x y b e n z o a t e h y d r o x y l a s e from P s e u d o m o n a s f l u o r e s c e n s W . J . H . v a n B e r k e l , F. M u l l e r , W.J. W e i j e r , P . A . J e k e l a n d J.J. B e i n t e m a 335 C h e m i c a l m o d i f i c a t i o n o f p h e n o l h y d r o x y l a s e b y p - n i t r o b e n z e n e - s u l p h o n y l f l u o r i d e C . T . S e j l i t z a n d H . V . N e u j a h r 335 S t u d i e s o f 2 , 5 - d i k e t o c a m p h a n e m o n o o x y g e n a s e from P s e u d o m o n a s p u t i d a A T C C 17453 D . G . T a y l o r a n d P . G . T r u d g i l l X V I I I Recent progress i n bioluminescence: c l o n i n g of the s t r u c t u r a l genes encoding b a c t e r i a l l u c i f e r a s e , a n a l y s i s of the encoded sequences, and c r y s t a l l i z a t i o n of the enzyme T.O. B a l d w i n , T . C . Johnston and R. Swanson 345 Probing the b a c t e r i a l l u c i f e r a s e aldehyde site by a f f i n i t y and photo a f f i n i t y l a b e l i n g S-C. Tu and A. F r i e d 359 The c a t a l y t i c turnover of b a c t e r i a l l u c i f e r a s e produces a q u a s i - s t a b l e species of altered conformation N.K. AbouKhair, M.M. Ziegler and T.O. B a l d w i n 371 Electron microscopy and X - r a y d i f f r a c t i o n studies on heavy r i b o f l a v i n synthase from B a c i l l u s s u b t i l i s R. Ladenstein, B. Meyer, R. Huber, H. L a b i s c h i n s k i , K. Bartels, H-D. B a r t u n i k , L. Bachmann, H.C. L u d w i g and A. Bacher 375 Heavy r i b o f l a v i n synthase from B a c i l l u s s u b t i l i s . Primary s t r u c t u r e and r e a g g r e g a t i o n of the B subunits H.L. L u d w i g , F. Lottspeich, A. Henschen, R . L a d e n s t e i n and A. Bacher 379 ENZYME R E A C T I O N MECHANISMS Mechanism of a , B-dehydrogenation of f a t t y a c i d CoA d e r i v a t i v e s by f l a v i n enzymes S. Ghisl a 385 B u t y r y l - C o A d e h y d r o g e n a s e : A s p e c t s o f a c c e p t o r and s u b s t r a t e s p e c i f i c i t y P . C . E n g e l , G. W i l l i a m s o n and L . Shaw 403 X I X O x y g e n r e a c t i v i t y of b u t y r y l - C o A d e h y d r o g e n a s e from M e g a s p h a e r a e l s d e n i i and from ox l i v e r m i t o c h o n d r i a P . A . E l l i s o n , L. S h a w , G. W i l l i a m s o n and P . C . Engel 4 1 3 S u i c i d e i n a c t i v a t i o n of s h o r t - c h a i n a c y l - C o A d e h y d r o g e n a s e s by propion y l - C o A . F o r m a t i o n of a s u b s t r a t e - f l a v i n a d d u c t L. Shaw and P . C . Engel 4 1 7 S t r u c t u r e of the f l a v i n N-5 a d d u c t free r a d i c a l o b t a i n e d f o l l o w i n g i n h i b i t i o n of s h o r t - c h a i n a c y l - C o A d e h y d r o g e n a s e by p r o p i o n y l - C o A G.N. George, L. S h a w , R . C . B r a y and P . C . Engel 421 P u r i f i c a t i o n and p r o p e r t i e s of f i v e d i s t i n c t a c y l - C o A d e h y d r o g e n a s e s from r a t l i v e r m i t o c h o n d r i a V. I k e d a , K.O. I k e d a , and K. T a n a k a 435 M e c h a n i s m of action of s h o r t - c h a i n , m e d i u m - c h a i n and l o n g - c h a i n a c y l - C o A d e h y d r o g e n a s e s i s o l a t e d from r a t l i v e r V. I k e d a , D. Hine, K.O. I k e d a and K. T a n a k a 4 39 I n a c t i v a t i o n of pig k i d n e y g e n e r a l a c y l - C o A d e h y d r o g e n a s e by 2 - a l k y n o y l - C o A d e r i v a t i v e s K. F r e u n d , J . P . Mizzer and C. Thorpe 4 4 3 On the i n a c t i v a t i o n of g e n e r a l a c y l - C o A d e h y d r o g e n a s e from p i g k i d n e y by m e t h y l e n e c y c l o p r o p y l - a c e t y l - C o A , a metabolite of h y p o g l y c i n H - D . Zeller and S. G h i s l a . 447 S t r u c t u r e - f u n c t i o n c o r r e l a t i o n i n B - o x i d a t i o n enzymes C. Rojas, W. G u s t a f s o n , J. S c h m i d t , D. D o m a n s k i , B. A. F e i n b e r g and J. T. M c F a r l a n d 451 X X P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of g l u t a r y l - C o A d e h y d r o g e n a s e , e l e c t r o n t r a n s f e r f l a v o p r o t e i n a n d ETF-CoQ o x i d o r e d u c t a s e from P a r a c o c c u s d e n i t r i f i c a n s M. H u s a i n and D.J. S t e e n k a m p 455 Reactions of ETF and ETF-CoQ o x i d o r e d u c t a s e D.J. S t e e n k a m p , R . R . R a m s a y and M. 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