Table Of ContentProceedings of the 11th FEBS Meeting
General Editor: Per Schambye, Odense
Volume 42 REGULATORY MECHANISMS OF
CARBOHYDRATE METABOLISM
Volume 43 GENE EXPRESSION
Volume 44 BIOCHEMICAL ASPECTS OF
NEW PROTEIN FOOD
Volume 45 MEMBRANE PROTEINS
Volume 46 REGULATION OF FATTY ACID AND
GLYCEROLIPID METABOLISM
Volume 47 REGULATORY PROTEOLYTIC
ENZYMES AND THEIR INHIBITORS
Volume 48 GROWTH FACTORS
Volume 49 FUNCTIONS OF ALTERNATIVE
TERMINAL OXIDASES
Volume 50 ALBUMIN
STRUCTURE, BIOSYNTHESIS, FUNCTION
FEBS Federation of European
Biochemical Societies
1 lth Meeting Copenhagen 1977
VOLUME 49
Colloquium B6
FUNCTIONS OF ALTERNATIVE
TERMINAL OXIDASES
Editors
HANS DEGN.
Odense
DAVID LLOYD, Cardiff
GEORGE C. HILL.
Fort Collins, Colorado
PERGAMON PRESS
OXFORD · NEW YORK · TORONTO · SYDNEY · PARIS · FRANKFURT
U.K. Pergamon Press Ltd., Headington Hill Hall,
Oxford OX3 OBW, England
U.S.A. Pergamon Press Inc., Maxwell House, Fairview Park,
Elmsford, New York 10523, U.S.A.
CANADA Pergamon of Canada Ltd., 75 The East Mall,
Toronto, Ontario, Canada
AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., 19a Boundary Street,
Rushcutters Bay, N.S.W. 2011, Australia
FRANCE Pergamon Press SARL, 24 rue des Ecoles,
75240 Paris, Cedex 05, France
FEDERAL REPUBLIC Pergamon Press GmbH, 6242 Kronberg-Taunus,
OF GERMANY Pferdstrasse 1, Federal Republic of Germany
Copyright © 1978 Pergamon Press Ltd.
All Rights Reserved. No part of this publication may be reproduced, stored in a
retrieval system or transmitted in any form or by any means: electronic, electrostatic,
magnetic tape, mechanical, photocopying, recording or otherwise, without
permission in writing from the publishers.
First edition 1978
Library of Congress Cataloging in Publication Data
Federation of European Biochemical Societies.
Meeting, 11th, Copenhagen, 1977
Functions of alternative terminal oxidases.
— (Publications; vol. 49).
1. Oxidases — Congresses
I. Title II. Schambye, Per III. Degn,
Hans IV. Lloyd, David V. Hill, George C
574. Γ 9248 QP603.08 77-30608
ISBN 0-08-022630-2
ISBN 0-08-021527-0 Setof9vols
In order to make this volume available as economically and as rapidly as possible the
authors' typescripts have been reproduced in their original forms. This method
unfortunately has its typographical limitations but it is hoped that they in no way
distract the reader.
Printed in Great Britain by William Clowes & Sons Limited
London, Beccles and Colchester
GENERAL INTRODUCTION TO THE PROCEEDINGS
The 11th FEBS Meeting, Copenhagen 1977,
was attended by more than 2500 biochemists and
their associates. More than 1300 posters, which
attracted many spectators and discussants, and
about 220 lectures constituted the back-bone of
the Meeting. It proved possible to run specia
list-sessions on grand topics in five days' sym
posia as well as colloquia-sessions treating
more limited problems.
We hope that the lectures from all six
symposia and three of the colloquia published
in the Proceedings volumes will be as supportive
to our science as they were to the substance of
the Meeting.
We are grateful for all the cooperative
efforts, in spite of the fact that the work had
to be done against deadlines, and also for the
support from the Publisher.
Per Schambye
Secretary-General
Professor of Biochemistry
Odense University
vii
ELECTRON TRANSPORT PATHWAYS ALTERNATIVE TO THE MAIN
PHOSPHORYLATING RESPIRATORY CHAIN
David Lloyd and Steven W. Edwards
Department of Microbiology, University College,
Cardiff, Wales
INTRODUCTION
A great diversity of cell-types, from bacteria to higher eukaryotic cells,
exhibit some capacity for respiration even when inhibitors of the main
phosphorylating respiratory chain are present at concentrations adequate to
prevent electron transport by this route. The alternative pathways
implicated in bacteria have been reviewed by Jurtshuk et d l (1975), Jones
(1977), and by Haddock & Jones (1977); those in the mitochondria of
eukaryotic microorganisms and in higher plants and animals have been
surveyed by Lloyd (1974), Henry & Nyns (1975) and by Solomos (1977). The
present review highlights the historical development of the field, outlines
the widespread occurrence of the phenomenon, traces the changing hypotheses
for mechanisms, hints at physiological functions and lists some unsolved
problems.
HISTORICAL DEVELOPMENT
Table 1 lists some important historical landmarks in the elucidation of
alternative pathways of electron transport; for a fascinating account of
early achievements, see Keilin (1966).
TABLE 1 Historical Landmarks
The respiration of ChZovelta is cyanide-resistant Warburg, 1919
Effects of cyanide on cytochromes in yeast, animals
and plants studied spectroscopically Keilin, 1925
Inhibition by CO depends on ratio CO/O2 and is light Warburg, 1926
sensitive Haldane, 1927
Keilin, 1927
Photochemical action spectrum of light-relief of Warburg &
CO-inhibited respiration of tövula utilis Negelein, 1928
Photochemical action spectrum of Aoetobactev Warburg &
pasteurianum Negelein, 1929
Cytochrome au> (d.) discovered in bacteria Yaoi & Tamiya, 1928
Modification of cytochrome spectrum of Aspergillus
ovyzae when surface mycelium becomes submerged
and grows Tamiya, 1928
1
D. Lloyd and S.W. Edwards
The respiration of sweet pea is cyanide-resistant Genevois, 1929
High resolution photochemical action spectrum of Kubowitz & Hass,
baker's yeast at 0.2°C 1932
Low sensitivity of CO-inhibited respiration of Negelein & Gerischer,
Azotobacter chrooooooum to relief by light 1934
Classification of bacterial respiration into those
stimulated by cyanide and CO, those inhibited
by cyanide but not CO, and those inhibited by
both (and subdivision of light-sensitive and
light-insensitive CO inhibition) Yamagutchi, 1934
Flavoprotein oxidases implicated in cyanide-
insensitive respiration of higher plants Van Herk, 1937
Characterization of cytochrome £3 by its reaction Keilin & Hartree,
with cyanide and CO 1938
A branch at the substrate side of cytochrome c
mediates cyanide-resistant respiration Okunuki, 1939
Unusual a-type cytochromes reported in Baker & Baumberger,
Tetrahymena 1941
The respiration of some protozoa is cyanide- Clark, 1945;
resistant Pace, 1945; Boer, 1945
Respiratory-deficient mutants of yeast Slonimski & Ephrussi,
characterized 1949
The respiration of anaerobically-grown yeast is Chin, 1950; Ephrussi
cyanide-resistant : changes on aerobic & Slonimski, 1950
adaptation
Mycelia of Myrotheoiwn verrucaria are cyanide- Darby & Goddard,
insensitive 1950
Respiratory-deficient mutants of Neurospora
characterized Mitchell et dl., 1953
"CO-binding pigment" described in bacteria Chance, 1953;
Smith, 1953
Cyanide-insensitive mitochondria isolated from
Arum spadix James & Elliott, 1955
Failure to detect cytochrome a aß in some
bloodstream forms of tryplfno somes and parasitic
anaerobic protozoa (Trichomonas sp.) Ryley, 1955; 1956
Cytochromes a^ and the protohaemin-like 'CO-binding Castor & Chance,
pigment' are functional oxidases in some 1955
bacteria as shown by photochemical action spectra
Proposal that cytochrome "bj" mediates cyanide-
insensitive respiration of Ariwispadix Bendall & Hill, 1956
Respiration of Avum spadix has high O2 affinity Yocum & Hackett, 1957
Cytochrome d.^£ characterized in Pseudomonas
aeruginosa Horio, 1958
G.R. Williams discovers cytochrome P-450 in 1955 Cooper et dl., 1965
2
Electron transport pathways
Photochemical characterization of cytochrome a«
(d) , "CO-binding pigment" renamed
cytochrome "jo" Castor & Chance, 1959
Evidence for "excess oxidase" hypothesis in
skunk cabbage mitochondria Chance & Hackett, 1959
Cytochrome £ peroxidase may by-pass Site III Yonetani & Ohnishi,
in yeast mitochondria 1966
H2O2 formation in mammalian mitochondria is on
the substrate side of cytochrome b^ Hinckle et al., 1967
Alternative pathway in plant mitochondria
branches in the» F -cytochrome b-UQ region Storey & Bahr, 1969
AMP-stimulated inducible alternative oxidase Sharpless & Butow,
described in Eugtena graoilis mitochondria 1970
Cyanide-insensitive respiration in plant
mitochondria does not involve cytochromes
but is mediated by a non-haem iron protein Bendall & Bonner, 1971
Hydroxamic acids inhibit the cyanide-insensitive
pathway Schonbaum et dl., 1971
Cytochrome £^90 ^ut not cytochrome o) is a Turner et al,9 1971;
terminal oxidase in Tetvahymena mitochondria Lloyd & Chance, 1972
Cyanide-insensitive respiration in poky mutants
of Neurospora does not involve cytochromes Lambowitz et al.-9 1972
Studies of control of relative electron fluxes
at the branch-point in plant mitochondria Bahr & Bonner, 1973
Cytochrome "o" is not a terminal oxidase in the Kusel & Storey, 1973;
trypanosome, Cvithidia fasc-iculata Edwards & Lloyd, 1973
Cytochrome £ may function as alternative terminal
oxidase in some trypanosomes Kronick & Hill, 1974
Fp is the equilibrium partner at the branch-
ma point in plant mitochondria Storey, 1976
Mitchellfs protonmotive UQ cycle is closely
associated with the alternative oxidase
pathway in plant mitochondria Rich & Moore, 1976
Colloquium 'Functions of Alternative Terminal
Oxidases' at the 11th FEBS Meeting at
Copenhagen, 1977
Over recent years, the phenomenon of cyanide-insensitive respiration has
received increased attention,. Table 2 lists the eukaryotic micro
organisms in which cyanide-insensitive respiration has been observed; some
of the factors responsible for the increased frequencies of reports over
the last two decades will be discussed later.
3
D. Lloyd and S.W. Edwards
TABLE 2 Frequencies of Reports of Cyanide-insensitive
Respiration
Yeasts Algae Protozoa FFiu ngi
1919-1929 1
1930-1939 1
1940-1949 6
1950-1959 2 11
1960-1969 18 4
1970-1977 9 1 6
PROPOSED MECHANISMS
At the beginning of this decade four different hypotheses had been proposed
to explain the mechanism of cyanide-resistant respiration in plant
mitochondria. Bendall & Bonner (1971) presented evidence against each of
these; it became clear that the process did not depend on low-affinity
flavoprotein oxidase, an alternative a-type cytochrome, or cytochrome b-^;
neither was it a question of incomplete inhibition of cytochrome c_ oxidase
by cyanide. Instead these investigators showed that the respiratory chain
bifurcates on the substrate side of the antimycin A-sensitive site, and
selective inhibition of the alternative oxidase pathway by metal complexing
agents (thiocyanate, a, a -dipyridyl and 8-hydroxyquinoline) suggested the
possible involvement of a non-haem iron protein. The discovery of more
specific inhibition of the alternative oxidase by hydroxamic acids (at
concentrations which do not inhibit or uncouple the main phosphorylating
respiratory chain) was a major break-through (Schonbaum et al9. 1971)
which together with the application of increasingly powerful e.p.r.
techniques swiftly led to the present exciting phase of rapid development
of this field of study, not only in plants but also in many eukaryotic
microorganisms.
Although the exact identity of the alternative oxidase remains elusive,recent
developments have led to the proposal of mechanisms at the branch-point
which are able to explain the behaviour of redox components under different
conditions. It is agreed that ubiquinone is the carrier common to both
the cytochrome and alternative oxidase pathways. Storey (1976) suggests
that ubiquinone is linked to the alternative oxidase by a flavoprotein (of
midpoint potential 50 millivolts more negative than the quinone with which
it is in equilibrium (Scheme 1). This arrangement can provide a switch
for the apportionment of electron transport; primarily through the
cytochrome chain under State 3 conditions, but through the alternate
oxidase when mitochondrial respiration is ADP limited.
A different mechanism based on Mitchellfs (1975) protonmotive ubiquinone
cycle has been suggested by Rich & Moore (1976) who envisage the specific
location of the branch-point of electron flow into the alternate pathway
at the reversal of the step of succinate dehydrogenase reduction of
QH* to QH (Scheme 2).
2
4
Electron transport pathways
Succinac NADU exogenous
/
Succinate NADH-dehydvogenase
dehydvogensss/. ' (external)
\ if-
NADH > NADH-dehydrogensse^--' F £"-«· Q ί=ϊ F > Cytochromes
(endogenous) (internal) 'la *.] ha ft
(Fe-S)? it Atitimycin A
F inhibition
x VIA
altor-iati? pa thw'ay
lx-->0...
WAW
Scheme 1
b_-566
M-side H* A%nt i HgSO\ SHAM[0 ] KCN H20
Scheme 2
Any demonstration of a cytochrome as a terminal oxidase must include both
kinetic and photochemical action data. There have been many claims that
cytochrome £ functions in eukaryotic systems as an alternative oxidase.
Conclusive evidence to the contrary has been obtained for the trypanosome
Crithidia fasoioulata (Edwards & Lloyd, 1973; Kusel & Storey, 1973) and
the former workers list five possible candidates for the identity of the
cytochrome £-like component of CO-difference spectra. Similarly
mitochondria from plants (Plesnicar et al. 1967), Tetrahymena (Lloyd &
Chance, 1972; Kilpatrick & Erecinska, 1977) and Acanthamoeba (Edwards et al.
1977) do not possess a functional cytochrome o, although all these cell
types have CO-binding pigments with spectral characteristics similar to
the bacterial cytochrome £. In many cases these have been shown to be
extra-mitochondrial in location. Photochemical action spectra have
however produced some evidence for cytochrome £ in Trypanosoma mega3
Blastocrithidia culicis and Leishmania taventolae (Kronick & Hill, 1974).
5
D. Lloyd and S.W; Edwards
PHYSIOLOGICAL FUNCTION
In some alternative pathways e.g. in bacteria where the terminal oxidases
are identifiable as cytochromes (Haddock & Jones, 1977), the efficiency of
coupling of phosphorylation to electron transport is identical to that of
the main chain. However what is intriguing is the physiological function
of alternative pathways which have little or no phosphorylating ability.
In systems where the main chain is impaired so that it has lowered or
negligible capacity, an alternative pathway which can account for excess
electron flux would seem logical. Micro-organisms have helped in the
understanding of the processes occurring in higher organisms because
alternative systems can be manipulated in the following ways:
1. Growth at different oxygen tensions e.g. Sacchavomyces cevevisiae
(Ephrussi & Slonimski, 1950).
2. Growth in the presence of inhibitors of macromolecular syntheses such as
chloramphenicol or ethidium bromide or inhibitors of electron transport(e.^.
in Euglena, Sharpless & Butow, 1970 and in Neuvospova cvassa, Lambowitz
et al.y 1972).
2+ =
3. Specific nutrient limitation e.g. Cu or SO/ limitation in Candida
utilis (Haddock & Garland, 1971; Downie & Garland, 1973), choline
deficiency in N. cvassa (Luck, 1965) or growth with different carbon sources
(Haddock & Jones, 1977). Also, the transition to the stationary phase of
growth is often accompanied by the development of alternative systems
(Akimenko & Medentzev, 1976).
4. Selection of mutants with altered respiratory components (e.g. poky
mutants in N. cvassa, Lambowitz et al.3 1972; von Jagow et αΙ.Λ 1973).
Reasons for the occurrence of alternative pathways, in preference to, or
alongside conventional phosphorylating chains, apart from the reasons
listed above, are more obscure but possible functions can be proposed:
1. Many organisms e.g. pseudomonads (Castric, 1975), Chlovella (Gewitz
et at., 1974) and many fungi and higher plants (Knowles, 1976) are able to
produce cyanide as a secondary metabolite. In fact, any organism capable
of producing a compound which can act as a respiratory chain inhibitor
might itself be expected to have a respiratory pathway which is insensitive
to that compound.
2. Some species, particularly the snow mould basidiomycetes (Strobel, 1964)
are capable of cyanide assimilation or utilization of the compound as sole
carbon or nitrogen source.
3. The mitochondrial cyanide-insensitive respiration of the Avum spadix
has been shown to be important in the generation of heat (Meeuse ,1975).
4. Where alternative pathways have reduced phosphorylating capacity, these
may function to regenerate oxidized coenzymes in the absence of
unnecessary ATP generation (Palmer, 1976).
5. Alternative oxidases which have oxygen affinities different from that of
the main chain may be important for growth at certain physiological oxygen
tensions. Values reported for apparent I^s for 0o vary widely; e.g.
Rhodotovula 23 uM (Matsunaka et at., 1966), Chlovella 6.7 μΜ (Sargent &
Taylor, 1972), Euglena 3.7 μΜ, (Sharpless & Butbw, 1970), Cu2 -limited
Candida 0.1 μΜ (Haddock & Garland, 1971).
6
