Proceedings 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