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Gas Enzymology: Proceedings of a Symposium held at Odense University, Denmark, 28–29 May 1984 PDF

252 Pages·1985·7.995 MB·English
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Gas Enzymology Gas Enzymology Proceedings ofa Symposium held at Odense University, Denmark, 28-29 May 1984 edited by H.Degn R.P. COX H. Toftlund Institutes of Biochemistry and Chemistry, Odense University, Odense, Denmark D. Reidel Publishing Company A MEMBER OF THE KLUWER ACADEMIC PUBLISHERS GROUP Dordrecht / Boston / Lancaster library of Congress Cataloging in Publication Data Main entry under title: Gas enzymology. Includes index. 1. Enzymes-Congresses. 2. Gas-Congresses. I. Degn, H. II. Cox, R. P. III. Toftlund, H. QP601.G335 1985 574.19'25 84-26230 ISBN-13:978-94-010-8831-2 e-ISBN-13:978-94-009-5279-9 DOl: I 0.1 007/978-94-009-5279-9 Published by D. Reidel Publishing Company, P.O. Box 17, 3300 AA Dordrecht, Holland Sold and distributed in the U.S.A. and Canada by Kluwer Academic Publishers, 190 Old Derby Street, Hingham, MA 02043, U.S.A. In all other countries, sold and distributed by Kluwer Academic Publishers Group, P.O. Box 322, 3300 AH Dordrecht, Holland All Rights Reserved © 1985 by D. Reidel Publishing Company, Dordrecht, Holland Softcover reprint of the hardcover 1st edition 1985 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner. CONTENTS PREFACE V11 CONTRIBUTORS 1X S. Bohatka QUADRUPOLE MASS SPECTROMETRIC MEASUREMENTS OF DISSOLVED AND FREE GASES 1 Y.M. Berlier, B. Dimon, G. Fauque and P.A. Lespinat DIRECT MASS-SPECTROMETRIC MONITORING OF THE METABOLISM AND ISOTOPE EXCHANGE IN ENZYMIC AND MICROBIOLOGICAL INVESTIGATIONS 17 D. Lloyd SIMULTANEOUS DISSOLVED OXYGEN AND REDOX MEASUREMENTS: USE OF POLAROGRAPHIC, BIOLUMINESCENCE AND MASS SPECTROMETRIC MONITORING COMBINED WITH DUAL-WAVELENGTH SPECTROPHOTOMETRY 37 D. Hill and D. Lloyd ALCOHOL OXIDASE IN CANDIDA BOIDINII, COMBINED SPECTRO PHOTOMETRIC AND OXYGEN MEASUREMENTS DURING ETHANOL INACTIVATION 55 O. Farver MECHANISM OF ACTIVATION AND REDUCTION OF DIOXYGEN BY RHUS LACCASE - A BLUE COPPER OXIDASE 61 B. Reinhammar DIOXYGEN REDUCTION IN BLUE OXIDASES: THE ELECTRON TRANSFER AND PROTONATION STEPS 79 R. Radmer and o. Ollinger PHOTOSYNTHESIS STUDIES USING MASS SPECTROMETRIC TECHNIQUES 91 A.J. Keys, N.P. Hall, M.A.J. Parry, C.N.G. Schmidt and S. Gutteridge INTERACTIONS OF CARBON DIOXIDE AND OXYGEN ON D-RIBULOSE 1,5-BISPHOSPHATE CARBOXYLATION 105 vi CONTENTS S. Lindskog THE CATALYTIC MECHANISM OF CARBONIC ANHYDRASE 121 D.N. Silverman, C.K. Tu and G.C. Wynns THE CATALYTIC MECHANISM OF CARBONIC ANHYDRASE STUDIED BY 180 EXCHANGE l35 O. Meyer and K. Fiebig ENZYMES OXIDIZING CARBON MONOXIDE 147 H. Dalton and D.J. Leak MECHANISTIC STUDIES ON THE MODE OF ACTION OF METHANE MONOOXYGENASE 169 L. Joergensen THE REACTION MECHANISM OF METHANE MONOOXYGENASE STUDIED BY MEMBRANE-INLET MASS SPECTROMETRY IN WHOLE CELLS OF METHANOTROPHIC BACTERIA 187 K. Hillman, D. Lloyd and A.G. Williams CONTINUOUS MONITORING OF FERMENTATION GASES IN AN ARTIFICIAL RUMEN SYSTEM (RUSITEC) USING A MEMBRANE-INLET PROBE ON A PORTABLE QUADRUPOLE MASS SPECTROMETER 201 B.B. Jensen ISOTOPE RATIO MASS-SPECTROMETRY STUDIES OF HD FORMATION BY NITROGENASE 207 G.J. Leigh and J.R. Postgate BINDING AND ACTIVATION OF DINITROGEN IN NITROGENASE 229 SUBJECT INDEX 247 PREFACE Being small, shapeless and inert a gas molecule does not seem to be an enzyme's dream of a substrate. Nevertheless evolution has provided a host of enzymes which can interact specifically with gas molecules such as oxygen, carbon dioxide, nitrogen, hydrogen etc. Many of these enzymes play dominant roles on the world scene in biogeochemical cycles. On the cellular level they tend to be closely connected to the energy conserving apparatus. We define Gas Enzymology as the study of these enzymes. Historically, Gas Enzymology is a subspecialty of bioenergetics. Its foundations, technical as well as conceptual were laid by Warburg in his studies of the cellular combustion of nutrients. The Warburg apparatus supported the first thirty years of research in the field. It was succeeded by the Clark electrode which had its heyday during the period when the modern concepts of bioenergetics took shape. The Clark electrode, itself approaching thirty years of age, is now being sup plemented and in some cases replaced by the vastly more powerful membrane inlet mass spectrometer which measures with equal ease all dis solved gases of interest in biochemistry. It is our belief that future development of Gas Enzymology will be linked to the widespread exploit ation of this technique. Beside the opening up of a wide range of new experimental possi bilities by the membrane inlet mass spectrometer, recent years have brought great progress in the elucidation of the catalytic sites of enzymes interacting with gas molecules. The possibility of using the enzymes as models for synthetic c~alysts for gas reactions is motiv ating an expanding field of research in chemical laboratories. At the same time enzyme processes catalyzing microbial gas exchanges have attained great interest from a biotechnological viewpoint. Having observed these interesting developments in various fields of Gas Enzymology and also pursued our own research in some of its fields for several years we felt a desire to see a unified picture. We decided to organize what we believe to be the first international sym posium on Gas Enzymology. The symposium was held at Odense University, Denmark in May 1984. The present book is its proceedings. In the selec tion of contributors we attempted to cover all main subjects within the field. Unavoidably there are omissions due to our economical limitations and oversights due to our ignorance. We apologize for such shortcomings and hope that they can be made up for in later symposia. The symposium on Gas Enzymology was supported by the Danish Natural Science Research Council (grant no. 11-4524), Ingeni~r N. Knudsens Fond and the Biophysical Society at Odense University. The editors CONTRIBUTORS Y.M. Berlier, Eguipe Commune d'Enzymologie CNRS/CEA, ARBS CEN-Cadarache, B.P. n 1, F-13115 Saint Paul lez Durance, France S. Bohatka, Institute of Nuclear Research of the Hungarian Academy of Sciences, Bern ter 18/C, H-4001 Debrecen Pf. 51, Hungary H. Dalton, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. B. Dimon, Equipe Commune d'Enzymologie CNRS/CEA, ARBS CEN-Cadarache, B.P. nO 1, F-13115 Saint Paul lez Durance, France O. Farver, Institute of Chemistry AD, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen 0, Denmark G. Fauque, Equipe Commune d'Enzymologie CNRS/CEA, ARBS CEN-Cadarache, B.P. nO 1, F-13115 Saint Paul lez Durance, France K. Fiebig, Institut fur Mikrobiologie der Georg-August-Universitat, Grisebachstrasse 8, D-3400 Gottingen, Federal Republic of Germany S. Gutteridge, Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. N.P. Hall, Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. D. Hill, Department of Microbiology, University College, Newport Road, Cardiff CF2 ITA, Wales, U.K. K. Hillman, Department of Microbiology, University College, Newport Road, Cardiff CF2 ITA, Wales, U.K. B.B. Jensen, Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark L. Joergensen, Institute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark A.J. Keys, Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. D.J. Leak, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. G.J. Leigh, AFRC Unit of Nitrogen Fixation, University of Sussex, Brighton BNl 9RQ, U.K. P.A. Lespinat, Equipe Commune d'Enzymologie CNRS/CEA, ARBS CEN-Cadarache, B.P. nO 1, F-13115 Saint Paul lez Durance, France S. Lindskog, Department of Biochemistry, University of Umea, S-90187 Umea, Sweden D. Lloyd, Department of Microbiology, University College, Newport Road, Cardiff CF2 ITA, Wales, U.K. O. Meyer, Institut fur Mikrobiologie der Georg-August-Universitat, Grisebachstrasse 8, D-3400 Gottingen, Federal Republic of Germany O. Ollinger, Biosciences Department, Martin Marietta Laboratories, 1450 South Rolling Road, Baltimore, MD 21227, USA x CONTRIBUTORS M.A.J. Parry, Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. J.R. Postgate, AFRC Unit of Nitrogen Fixation, University of Sussex, Brighton BNl 9RQ, U.K. R. Radmer, Biosciences Department, Martin Marietta Laboratories, 1450 South Rolling Road, Baltimore, MD 21227, USA B. Reinhammar, Department of Biochemistry and Biophysics, University of Goteborg and Chalmers Institute of Technology, S-4l296 Goteborg, Sweden C.N.G. Schmidt, Department of Biochemistry, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. D.N. Silverman, Department of Pharmacology, University of Florida, College of Medicine, Gainesville, Florida 32610, USA C.K. Tu, Department of Pharmacology, University of Florida, College of Medicine, Gainesville, Florida 32610, USA A.G. Williams, Hannah Research Institute, St. Quivox, Ayr KA6 5HL, U.K. G.C. Wynns, Department of Pharmacology, University of Florida, College of Medicine, Tainesville, Florida 32610, USA QUADRUPOLE MASS SPECTROMETRIC MEASUREMENT OF DISSOLVED AND FREE GASES Sandor Bohatka Institute of Nuclear Research of the Hungarian Academy of Sciences (ATOMKI) Bern ter 18/c H-4001 Debrecen Pf.SI Hungary ABSTRACT. A quadrupole mass spectrometer (QMS) system was constructed for the measurement of fermentation gases. This work is based on the experience in constructing QMS, respiratory and blood-gas analysers. The analyser system shown here has a microcomputer controlled QMS for multi component analysis and a multi-channel sampling unit. The latter makes possible static and dinamic sampling o:f gases exhausted from the fermentor and gases dissolved in the fermentation broth. Examples are shown for its capabili ties for on-line monitoring of long-term fermentation processes and off-line analysis of some components which were made volatile by adequate chemical treatments. The QMS equipment is a substantial part of a monitor system and the process control of a pilot plant of lSOO~ fer mentors. It is capable of monitoring various fermentors quasi-simultaneously in industrial circumstances. Similar QMS technique was used for the in vivo measurement of free and dissolved gases of plants. 1. INTRODUCTION There are also gases among the nutrients and metabolic products of living creatures. It is inevitable to measure these gas components when the behaviour of biological sys tems must be determined. Organisms generally contain much water, many of them live only in water, therefore gases must be measured not only in gas phase but in dissolved state, too. This is a basic question in cell cultures, in plant-, animal- and human tissues. Mass spectrometers (MS) have the possibility of mUlticomponent analysis of free and dissolved gases. They offer non-destructive sampling methods which can be applied to in vivo and in situ measurements. The medical research and diaRnostics gave an impetus H. Degn et al. (eds.), Gas Enzymology, 1-16. © 1985 by D. Reidel Publishing Company. 2 s. BOHATKA to this technique. It has been recognized first in res piratory gas analysis that mass spectrometry is one of the few methods which are able to make mUlticomponent analysis [1-3]. Most of the others are specific: one instrument can measure only one component or a definite group of compo nents (e.g. paramagnetic, infra-red absorption). Gas-chromatograph is almost as effective as MS but it is much slower. Since the 1960s mobile, sensitive multichannel MS systems have been constructed for respiratory analysis (Varian: Atlas M-3, Medispec: MS-4, MS-8, Godart: RMS-36 , Pelkin-Elmer: MGA 1100, Centronics: MGA-200, Medicor: QGA- 11, etc.), and they are the most effective instruments in medicine for the analysis of free gases. The expired air provides many information about the health of the patient but the primary processes take place mostly in liquid phase. Gases dissolved in blood and tissues must be meas ured if a complete picture is to be drawn [4,5]. This need led to the construction of mass-spectrometric blood-gas analysers. Only their price and the invasive sampling technique hindered their general use but it was early re cognized that the instruments of blood-gas analysis could be applied easily and favourably in other fields, too. The membrane inlet mass spectrometry is a non-invasive method in the study of cell suspensions, fermentation liquids and plants, and it provides a basic contribution to the com plex analysis of biological systems [6-18]. A mass spectrometer coupled to a membrane inlet can detect free and physically dissolved gases directly, and the measurement of other non-volatile or chemically dis solved components is also possible if a volatile component can be produced through some chemical or enzyme-catalyzed reaction [7]. Most of the papers report on experiments where the volume of liquid was in the range of 1 mt-100t • The aim of the joint research program of BIOGAL Pharma ceutical Works and ATOMKI has been to measure gases in large scale fermentors in industrial circumstances. This paper gives an account on the instrumentation and method of mass spectrometric analysis of fermentation gases. An other application of MS-technique: plant-physio logical measurements - an independent project in ATOMKI and some biological and agricultural research centres - is closely connected with the former one in one respect: the materials and methods. 2. MATERIALS AND METHODS 2.1. Instruments The samples to be analysed have to be transported to the mass spectrometer, and their signals in the MS must exceed that of the background. Fairly sophisticated sampling and

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