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

252 Pages·1985·7.995 MB·English

by  Sándor Bohátka (auth.), H. Degn, R. P. Cox, H. Toftlund (eds.)

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
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© 1985 by D. Reidel Publishing Company, Dordrecht, Holland
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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