New comprehensive biochemistry vol 23 molecular mechanisms in bioenergetics

MOLECULAR MECHANISMS
IN BIOENERGETICS


New Comprehensive Biochemistry

Volume 23

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Utrecht

ELSEVIER
Amsterdam . London. New York . Tokyo


Molecular Mechanisms
in Bioenergetics

Editor

LARS ERNSTER
Department of Biochemistry, Arrhenius Luhorutories for Nutural Sciences,
Stockholm University, S-106 91 Stockholm, Sweden

1992

ELSEVIER
Amsterdam . London . New York . Tokyo


ELSEVIER SCIENCE B.V.
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First edition 1992
Second impression 1994
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Q

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Printed in The Netherlands


V

Introduction
‘Researchis to see what everybody has seen nnd think whnt nobody hns thought’
Albert Szent-Gyorgyi: Bioenergetics
(Academic Press, New York, 1957)

Bioenergetics is the study of energy transformations in living matter. It is now well established
that the cell is the smallest biological entity capable of handling energy. Every living cell has the
ability, by means of suitable catalysts, to derive energy from its environment, to convert it into
a biologically useful form, and to utilize it for driving life processes that require energy. In
recent years, research in bioenergetics has increasingly been focused on the first two of these
three aspects, i.e., the reactions involved in the capture and conversion of energy by living cells,
in particular those taking place in the energy-transducing membranes of mitochondria, chloroplasts and bacteria. This area, often referred to as membrane bioenergetics, has been the topic
of the volume on Bioenergetics published within this series in 1984 (volume 9). As pointed out
in the Introduction of that volume, important progress had just begun towards a merger
between bioenergetics and molecular biology, i.e., a transition of membrane bioenenergetics
into molecular bioenergetics. This progress has now reached the stage where the publication of
a volume on Molecular Mechanisms in Bioenergetics was felt to be timely. As in the previous
volume, the purpose of this Introduction is to put these developments into a historical perspective. For details, the reader is referred to the large number of historical reviews on bioenergetics
that have appeared over the past years, a selection of which is listed after this Introduction.
Bioenergetics as a scientific discipline began a little over 200 years ago, with the discovery of
oxygen. Priestley’s classical observation that green plants produce and animals consume oxygen, and Lavoisier’s demonstration that oxygen consumption by animals leads to heat production, are generally regarded as the first scientific experiments in bioenergetics. At about the
same time Scheele, who discovered oxygen independently of Priestley, isolated the first organic

compounds from living organisms. These developments, together with the subsequent discovery by Ingen-Housz, Senebier and de Saussure that green plants under the influence of sunlight
take up carbon dioxide from the atmosphere in exchange for oxygen and convert it into organic
material, played an important role in the development of concepts leading to the enunciation
of the First Law of Thermodynamics by Mayer in 1842.
A recurrent theme in the history of bioenergetics is vitalism, i.e., the reference to ‘vital
forces’, beyond the reach of physics and chemistry, to explain the mechanism of life processes.
For about half a century following Scheele’s first isolation of organic material from animals
and plants it was believed that these compounds, which all contained carbon, could only be
formed by living organisms - hence the name organic - a view which, however, was not shared
by some chemists, e.g., Liebig and Wohler. Indeed, in 1828 Wohler succeeded for the first time
in synthesizing an organic compound, urea, in the laboratory. This breakthrough was soon
followed by other organic syntheses. Thus, the concept that only living organisms can produce
organic compounds could not be maintained.
At the same time, however, it became increasingly evident that living organisms could pro-


vi
duce these compounds better, more rapidly and with greater specificity, than could the chemist
in his test tube. The idea, first proposed by Berzelius in 1835, that living organisms contained
catalysts for carrying out their reactions, received increasing experimental support. Especially
the work of Pasteur in the 1860s o n fermentation by brewer’s yeast provided firm experimental
basis for the concept of biocatalysis. Pasteur’s work was also fundamental in showing that
fermentation was regulated by the accessibility of oxygen the ‘Pasteur effect’ - which was the
first demonstration of the regulation of energy metabolism in a living organism. In attempting
to explain this phenomenon Pasteur was strongly influenced by the cell theory developed in the
1830s by Schleiden and Schwann, according to which the cell is the common unit of life in
plants and animals. Pasteur postulated that fermentation by yeast required, in addition to a
complement of active catalysts - ‘ferments’ - also a force vitale that was provided by, and
dependent on, a n intact cell structure. This ‘vitalistic’ view was again strongly opposed by
Liebig, who maintained that it should be possible to obtain fermentation in a cell-free system.

This indeed was achieved in ‘1897 by Biichner, using a press-juice of yeast cells.
In the early 1900s important progress was made toward the understanding of the role of
phosphate in cellular energy metabolism. Following Biichner’s demonstration of cell-free fermentation, Harden and Young discovered that this process required the presence of inorganic
phosphate and a soluble, heat-stable cofactor which they called cozymase (later identified as
the coenzyme nicotinamide adenine dinucleotide). These discoveries opened the way to the
elucidation of the individual enzyme reactions and intermediates of glycolysis. The identification of various sugar phosphates by Harden and Young, Robison, Neuberg, Embden, Meyerhof, von Euler and others, and the clarification of the role of cozymase in the oxidation of
3-phosphoglyceraldehyde by Warburg are the most important landmarks of this development.
A milestone in the history of bioenergetics was the discovery of ATP and creatine phosphate
by Lohmann and by Fiske and Subbarow in 1929. Their pioneering findings that working
muscle splits creatine phosphate and that the creatine so formed can be rephosphorylated by
ATP, were followed in the late 1930s by Engelhardt’s and Szent-GyOrgyi’s fundamental discoveries concerning the role of ATP in muscle contraction. At about the same time Warburg
demonstrated that the oxidation of 3-phosphoglyceraldehyde is coupled to ATP synthesis and
Lipmann identified acetyl phosphate as the product of pyruvate oxidation in bacteria. In 1941,
Lipmann developed the concept of ‘phosphate-bond energy’ as a general principle for energy
transfer between energy-generating and energy-utilizing cellular processes. It seemed that it
was only a question of time until most of these processes could be reproduced and investigated
using isolated enzymes.
Parallel to these developments, however, vitalism re-entered the stage in connection with
studies of cell respiration. In 1912 Warburg reported that the respiratory activity of tissue
extracts was associated with insoluble cellular structures. He called these structures ‘grana’ and
suggested that their role is to enhance the activity of the iron-containing respiratory enzyme,
Atmungsjerment. Shorty thereafter Wieland, extending earlier observations by Battelli and
Stern, reached a similar conclusion regarding cellular dehydrogenases. Despite diverging views
concerning the nature of cell respiration - involving an activation of oxygen according to
Warburg and an activation of hydrogen according to Wieland - they both agreed that the role
of the cellular structure may be to enlarge the catalytic surface. Warburg referred t o the
’charcoal model’ and Wieland to the ‘platinum model’ in attempting t o explain how this may
be achieved.
In 1925 Keilin described the cytochromes, a discovery that led the way to the definition of
the respiratory chain as a sequence of redox catalysts comprising the dehydrogenases a t one

end and Atmungsferment at the other, thereby bridging the gap in opinion between Warburg
~


vii
and Wieland. Using a particulate preparation from mammalian heart muscle, Keilin and Hartree subsequently showed that Warburg’s Atmungsferment was identical to Keilin’s cytochrome u3. They recognized the need for a cellular structure for cytochrome activity, but
visualized that this structure may not be necessary for the activity of the individual catalysts,
but rather for facilitating their mutual accessibility and thereby the rates of interaction between
the different components of the respiratory chain. Such a function, according to Keilin and
Hartree, could be achieved by ‘unspecific colloidal surfaces’. Interestingly, the possible role of
phospholipids was not considered in these early studies and it was not until the 1950s that the
membranous nature of the Keilin-Hartree heart-muscle preparation and its mitochondria1
origin were recognized.
During the second half of the 1930s important progress was made in elucidating the reaction
pathways and energetics of aerobic metabolism. In 1937 Krebs formulated the citric acid cycle,
and the same year Kalckar presented his first observations leading to the demonstration of
aerobic phosphorylation, using a particulate system derived from kidney homogenates. Earlier, Engelhardt had obtained similar indications with intact pigeon erythrocytes. Extending
these observations, Belitser and Tsybakova concluded from experiments with minced muscle
in 1939 that at least two molecules of ATP are formed per atom of oxygen consumed. These
results suggested that phosphorylation probably occurs coupled to the respiratory chain. That
this was the case was further suggested by measurements reported in 1943 by Ochoa, who
deduced a P i 0 ratio of 3 for the aerobic oxidation of pyruvate in heart and brain homogenates.
In 1945 Lehninger demonstrated that a particulate fraction from rat liver catalyzed the oxidation of fatty acids, and in 1948-1949 Friedkin and Lehninger provided conclusive evidence for
the occurrence of respiratory chain-linked phosphorylation in this system using /?-hydroxybutyrate or reduced nicotinamide adenine dinucleotide as substrate.
Although mitochondria had been observed by cytologists since the 184Os, the elucidation of
their function had t o await the availability of a method for their isolation. Such a method,
based on fractionation of tissue homogenates by differential centrifugation, was developed by
Claude in the early 1940s. Using this method, Claude, Hogeboom and Hotchkiss concluded in
1946 that the mitochondrion is the exclusive site of cell respiration. Two years later this conclusion was further substantiated by Hogeboom, Schneider and Palade with well-preserved mitochondria isolated in a sucrose medium and identified by Janus Green staining. In 1949 Kennedy and Lehninger demonstrated that mitochondria are the site of the citric acid cycle, fatty
acid oxidation and oxidative phosphorylation.

In 1952- 1953 Palade and Sjostrand presented the first high-resolution electron micrographs
of mitochondria. These micrographs served as the basis for the now generally accepted notion
that mitochondria are surrounded by two membranes, a smooth outer membrane and a folded
inner membrane giving rise to the cristae. In the early 1950s evidence also began to accumulate
indicating that the inner membrane is the site of the respiratory-chain catalysts and the ATPsynthesizing system. In the following years research in many laboratories was focussed on the
mechanism of electron transport and oxidative phosphorylation, using both intact mitochondria and ‘submitochondrial particles’ consisting of vesiculated inner-membrane fragments.
Studies with intact mitochondria, performed in the laboratories of Boyer, Chance, Cohn,
Green, Hunter, Kielley, Klingenberg, Lardy, Lehninger, Lindberg, Lipmann, Racker, Slater
and others, provided information on problems such as the composition, kinetics and the localization of energy-coupling sites of the respiratory chain, the control of respiration by ATP
synthesis and its abolition by ‘uncouplers’, and various partial reactions of oxidative phosphorylation. Most of the results could be explained in terms of the occurrence of nonphosphorylated high-energy compounds as intermediates between electron transport and ATP


...

Vlll

synthesis, a chemical coupling mechanism envisaged by several laboratories and first formulated in general tems by Slater. However, intensive efforts to demonstrate the existence of such
intermediates proved unsuccessful.
Studies with beef-heart submitochondrial particles initiated in Green’s laboratory in the
mid-1950s resulted in the demonstration of ubiquinone and of non-heme iron proteins as
components of the electron-transport system, and the separation, characterisation and reconstitution of the four oxidoreductase complexes of the respiratory chain. In 1960 Racker and his
associates succeeded in isolating an ATPase from submitochondrial particles and demonstrated that this ATPase, called F,, could serve as a coupling factor capable of restoring
oxidative phosphorylation to F,-depleted particles. These preparations subsequently played an
important role in elucidating the role of the membrane in energy transduction between electron
transport and ATP synthesis.
A somewhat similar development took place concerning studies of the mechanism of photosynthesis. Although the existence of chloroplasts and their association with chlorophyll had
been known since the 1830s, and their identity as the site of carbon dioxide assimilation was
established in 1881 by Engelmann using isolated chloroplasts, it was not until the 1930s that the
mechanism of photosynthesis began to be clarified. In 1938 Hill demonstrated that isolated
chloroplasts evolve oxygen upon illumination, and beginning in 1945 Calvin and his associates

elucidated the pathways of the dark reactions of photosynthesis leading to the conversion of
carbon dioxide to carbohydrate.
The latter process was shown to require ATP, but the source of this ATP was unclear and a
matter of considerable dispute. The breakthrough came in 1954 when Arnon and his colleagues
demonstrated light-induced ATP synthesis in isolated chloroplasts. The same year Frenkel
described photophosphorylation in cell-free preparations of bacteria. Photophosphorylation
in both chloroplasts and bacteria was found to be associated with membranes, in the former
case with the thylakoid membrane and in the latter with structures derived from the plasma
membrane, called chromatophores. In the following years work in a number of laboratories,
including those of Arnon, Avron, Chance, Duysens, Hill, Jagendorf, Joliot, Kamen, Kok, San
Pietro, Trebst, Witt and others, resulted in the identification and characterization of various
catalytic components of photosynthetic electron transport. Chloroplasts and bacteria were
also shown to contain ATPases similar to the F,-ATPase of mitochondria.
By the beginning of the 1960s it was evident that both oxidative and photosynthetic phosphorylation were dependent on an intact membrane structure, and that this requirement probably was related to the interaction of the electron-transport and ATP-synthesizing systems
rather than the activity of the individual catalysts. However, contemporary thinking concerning the mechanism of ATP synthesis was dominated by the chemical coupling hypothesis and
did not readily envision a role for the membrane. This impasse was broken in 1961 when
Mitchell first presented his chemiosmotic hypothesis, according to which energy transfer between electron transport and ATP synthesis takes place by way of a transmembrane proton
gradient.
Mitchell’s hypothesis was first received with skepticism, but in the mid- 1960s evidence began
to accumulate in favour of the chemiosmotic coupling mechanism. It was shown that electrontransport complexes and ATPases, when present in either native or artificial membranes, are
capable of generating a transmembrane proton gradient and that this gradient can serve as the
driving force for electron transport-linked ATP synthesis. Agents that abolished the proton
gradient uncoupled electron transport from phosphorylation. Proton gradients were also
shown to be involved in various other membrane-associated energy-transfer reactions, such as
the energy-linked nicotinamide nucleotide transhydrogenase, the synthesis of inorganic pyro-


1x
phosphate, the active transport of ions and metabolites, mitochondrial thermogenesis in brown
adipose tissue, and light-driven ATP synthesis and ion transport in Halobacteria. In recent

years it has also been demonstrated that in several instances a sodium ion gradient, rather than
a proton-motive force, can serve as the electrochemical device in membrane-associated energy
transduction in connection with both electron transport and ATP synthesis. The chapters of
this volume give an overview of our present state of knowledge concerning these processes.
The major problems in this field that remain to be solved concern, on the one hand, the
topologic and dynamic aspects of membrane-associated energy-transducing catalysts at the
molecular level; and, on the other hand, the mechanisms responsible for the biosynthesis and
regulation of these catalysts in the intact cell and organism.
Although there is a great deal of information available today about the primary structure
and subunit composition of the various catalysts, knowledge of their tertiary structure and
membrane topology is still rather limited; in fact, the only example of a membrane-associated
energy-transducing enzyme complex whose structure is known at the atomic level of resolution
is the photosynthetic reaction center of purple bacteria. Also, relatively little is known about
the conformational events active-site rearrangements, protein-subunit and lipid-protein interactions - that take place during catalysis, and about the mechanisms by which these events
are linked to the translocation of protons or other charged species that are instrumental in
establishing the electrochemical gradients mediating energy transfer between the various catalysts. Indeed, there is not a single instance of precise knowledge about the mode of operation
of a protein involved in the translocation of protons or any other ions across a biological
membrane.
Regarding the biosynthesis and regulation of energy-transducing catalysts, important progress has been made over the last few years especially in the understanding of the role of the
mitochondrial and chloroplast genomes in the synthesis of various subunits of the energytransducing electron-transfer complexes and ATP synthase. Questions of great current interest
are the mechanisms by which the synthesis of these subunits is coordinated with that of their
nuclear-encoded counterparts and with the transport of the latter into the organelles. Another
still poorly understood problem is the function of the noncatalytic subunits of various energytransducing enzyme complexes, their variation in number and structure from one species or
organ to another, and their possible role in the regulation of the biosynthesis and assembly of
these complexes.
The chapters of this volume deal with some of the above problems, describing progress that
has been made during the last few years due to the development of new methods and concepts
within various disciplines, including biophysics, biochemistry, molecular and cell biology, genetics and pathophysiology. Due to these developments, we can foresee a continued rapid
progress in understanding the molecular details of cellular energy transduction. At the same
time, this progress has widened our perspective of bioenergetics, from molecules, membranes,

organelles and cells back to the organism as a whole, i.e. where the whole story began over two
centuries ago.
Before terminating this introduction it is a true pleasure to express my thanks to the authors
of the various chapters for having accepted the invitation to contribute to this volume and, in
particular, for their efforts to submit their manuscripts in time which has made it possible to
publish this volume while its contents are still reasonably up-to-date. I was deeply shocked and
saddened by the death of Peter Mitchell on April IOth, 1992. He had agreed to write a chapter
on ‘Chemiosmotic Molecular Mechanisms’ but was unable to complete it because of illness. I
am greatly indebted to Vladimir Skulachev for his willingness to extend his already submitted
~


X

chapter on “a+ Bioenergetics’ by including an introductory section on chemiosmotic systems
in general.
Finally I wish to thank my colleague Kerstin Nordenbrand at the Arrhenius Laboratories
for her valuable help with the editorial work, and the staff of Elsevier Science Publishers B.V.,
in particular the Acquisition Editor Amanda Shipperbottom, the Desk Editor Dirk de Heer
and the Promotion Manager Anthony Newman, for friendly and efficient cooperation.
Lars Ernster
Department of Biochemistry
Arrhenius Laboratories
Stockholm University
S-106 91 Stockholm
Sweden


x1

Some reviews on topics related to the history
of bioenergetics
Rabinowich, E.I. (1945) Photosynthesis and Related Processes. Interscience, New York.
Lindberg, 0. and Ernster, L. (1954) Chemistry and Physiology of Mitochondria and Microsomes. Springer, Vienna.
Krebs, H.A. and Kornberg, H.L. (1957) A survey of the energy transformations in living matter. Ergeb.
Physiol. 49, 212-298.
Novikoff, A.B. (1961) Mitochondria (Chondriosomes). In: The Cell, Vol. 11, pp. 299-421. Brachet, J. and
Mirsky, A.E (eds.) Academic Press, New York.
Lehninger. A.L. (1964) The Mitochondrion. Benjamin, New York.
Keilin, D. (1966) The History of Cell Respiration and Cytochrome. Cambridge University Press. Cambridge.
Slater, E.C. (1966) Oxidative Phosphorylation, Comprehensive Biochemistry, Vol. 14, pp. 327-396.
Elsevier, Amsterdam.
Kalckar, H.M. (1969) Biological Phosphorylations, Development of Concepts. Prentice-Hall, Englewood,
NJ.
Krebs, H.A. (1970) The history of the tricarboxylic acid cycle. Perspect. Biol. Med. 14. 154-170.
Wainio, W.W. (1970) The Mammalian Mitochondria1 Respiratory Chain. Academic Press, New York.
Lipmann, F. (1971) Wonderings of a Biochemist. Wiley-Interscience, New York.
Fruton, J.S. (1972) Molecules and Life. Wiley-Interscience, New York.
Arnon, D.I. (1977) Photosynthesis 1950- 1975, Changing concepts and perspectives. In: Photosynthesis I,
Trebst, A. and Avron, M. (eds.) Encyclopedia of Plant Physiology, New Series, Vol. 5, pp. 7-56.
Springer, Heidelberg.
Boyer, P.D., Chance, B., Ernster, L., Mitchell, P.. Racker, E. and Slater, E.C. (1977) Oxidative phosphorylation and photophosphorylation. Annu. Rev. Biochem. 46, 955-1026.
Racker, E. (1980) From Pasteur to Mitchell: A hundred years of bioenergetics. Fed. Proc. 39.210-215.
Bogorad, L. (1981) Chloroplasts. J. Cell Biol. 91, 256s 270s.
Ernster, L. and Schatz, G. (1981) Mitochondria: A historical review. J. Cell Biol. 91, 227s-255s.
Skulachev, V.P. (1981) The proton cycle: History and problems of the membrane-linked energy transduction, transmission, and buffering. In: Chemiosmotic Proton Circuits in Biological Membranes, pp.
3-46. Skulachev, V.P. and Hinkle, P.C. (eds.) Addison-Wesley, Reading, MA.
Slater, E.C. (1981) A short history of the biochemistry of mitochondria. In: Mitochondria and Microsomes, pp. 15-43. Lee. C.P., Schatz G . and Dallner, G . (eds.) Addison-Wesley, Reading, MA.
Tzagoloff, A. (1982) Mitochondria. Plenum Press, New York.
Hoober, J.K. (1984) Chloroplasts. Plenum Press, New York.

Ernster, L., ed. ( I 984) Bioenergetics. New Comprehensive Biochemistry, Vol. 9. Elsevier, Amsterdam.
Lee, C.P., ed. (1984, 1985,1987, 1991)Current Topics in Bioenergetics, Vols. 13- 16. Academic Press, New
York.
Quagliariello, E., Slater, E.C., Palmieri, F., Saccone, C. and Kroon, A.M., eds. (1985) Achievements and
Perspectives of Mitochondrial Research. Elsevier, Amsterdam.
Slater, E.C. (1987) Cytochrome systems: From discovery to present developments. In: Cytochrome Systems: Methods, Molecular Biology and Bioenergetics, pp. 3-1 1. Papa, S., Chance, B. and Ernster, L.
(eds.) Plenum Press, New York.
Ernster, L. and Lee, C.P. (1990) Thirty years of mitochondria1 pathophysiology: From Luft’s disease to
oxygen toxicity. In: Bioenergetics: Biochemistry, Molecular Biology, and Pathology, pp. 45 1-465. Kim,
C.H. and Ozawa, T. (eds.) Plenum Press, New York.
Barber, J., ed. (1992) The Photosystems: Structure, Function, Molecular Biology. Topics in Photosynthesis, Vol. 11. Elsevier, Amsterdam.


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xiii

List of contributors
B. Anderson, 121
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
G. Attardi, 483
Division of Biology, California Institute of Technology, Pasadena, P A 91 125, U.S.A.
H. Baltscheffsky, 331
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
M. Baltscheffsky, 331
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
G. Bechmann, 199
Institut fur Biochemie, Heinrich- Heine- Universitat DusseldorJ; UniversitatsstraJe I , W-4000
Dusseldorf I . Germany.

B. Cannon, 385
The Wenner-Gren Institute, Arrhenius Laboratories for Natural Sciences F3, Stockholm University, S-106 91 Stockholm, Sweden.
A. Chomyn, 483
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
G.B. Cox, 283
Membrane Biochemistry Group, Division of Biochemistry and Molecular Biology, John Curtin
School of Medical Research, Australian National University, Canberra, A. C. T. 2601, Australia.
R.L. Cross, 317
Department of Biochemistry & Molecular Biology, State University of New York, College of
Medicine, Health Science Center, 750 East Adams Street, Syracuse, N Y 13210, U.S.A.
J. Deisenhofer, 103
Howard Hughes Medical Institute Research Laboratories, University of Texas, Southwestern
Medical Center at Dallas, 5323 Harry Hines Blvd., Room Y4-206, Dallas, T X 75235-9050,
U.S.A.
R.J. Devenish, 283
Centre for Molecular Biology and Medicine, Department of Biochemistry, Monash University,
Clayton, Vic. 3168, Australia.
L. Ernster, v
Department of Biochemistry, Arrhenius Laboratories f o r Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
L.-G. Franzen, 121
Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
F. Gibson, 283
Membrane Biochemistry Group, Division of Biochemistry and Molecular Biology, John Curtin
School of Medical Research, Australian National University, Canberra, A . C. T. 2601, Australia.
T. Haltia, 217
Helsinski Bioenergetics Group, Department of Medical Chemistry, University of Helsinski.
Siltavuorenpenger IOA, SF-001 70 Helsinski, Finland.


xiv

Y. Hatefi, 265
Division of Biochemistry, Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, 10686 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
L. Hederstedt, 163
Department of Microbiology, University of Lund, Solvegatan 21, S-223 62 Lund, Sweden.
J.B. Hoek, 421
Department of Pathology and Cell Biology, Thomas Jefferson University, Rm. 271 JAH, 1020
Locust Street, Philadelphia, P A 19107, U.S.A.
S.M. Howitt, 283
Membrane Biochemistry Group, Division of Biochemistry and Molecular Biology, John Curtin
School of Medical Research, Australian National University, Canberra, A. C. T. 2601, Australia.
B. Kadenbach, 241
Fachbereich Chemie, Biochemie der Philipps- Universitat, Hans-Meerwein-Strape, 3550 Marburg, Germany.
R. Kramer, 359
Institut fur Biotechnologie I, Forschungszentrum Juelich, Juelich, Germany.
H. Michel, 103
Abteilung Molekulare Membranbiochemie, Max-Planck-Institut fur Biophysik, Frankfurt am
Main, Germany.
P. Nagley, 283
Centre for Molecular Biology and Medicine, Department of Biochemistry, Monash University,
Clayton, Vic. 3168, Australia.
J. Nedergaard, 385
The Wenner-Gren Institute, Arrhenius Laboratories for Natural Sciences F3, Stockholm University, S-106 91 Stockholm, Sweden.
T. Ohnishi, 163
Department of Biochemistry and Biophysics, University of Pennsylvania, A606 Richards Building, Philadelphia, PA 19104-6089, U.S.A.
F. Palmieri, 359
Department of Pharmaco-Biology, Laboratory of Biochemistry & Molecular Biology, University
ofBari, Trav. 200 Re David4, I-70125 Bari, Italy.
G.K. Radda, 463
M R C Biochemical and Clinical Magnetic Research Unit, Department osBiochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom.
R.R. Ramsay, 145

Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, C A 94121, U.S.A .
A. Reimann, 241
Fachbereich Chemie, Biochemie der Philipps- Universitat, Hans-Meerwein-Strape, 3550 Marburg, Germany.
R. Renthal, 75
Division of Earth & Physical Sciences, The University oj Texas, San Antonio, TX 78249, U.S.A.
C. Richter, 349
Laboratory of Biochemistry I, Swiss Federal Institute of Technology, UniversitatstraJe 16, CH8092 Zurich, Switzerland.
U. Schulte, 199
Institut fur Biochemie, Heinrich- Heine- Universitat Dusseldorf; Universitatsstraje I , W-4000
Dusseldorf 1, Germany.


xv
T.P. Singer, 145
Molecular Biology Division, 15l-S, Vetaruns Administration Medical Center, 4150 Clement
Street, San Francisco, CA 94121, U S.A
V.P. Skulachev, 37
Department of Bioenergetics, A . N . Belozersky Institute of Physico-Chemical Biology, Moscocv
State University, Moscow 119899, Russia.
D.J. Taylor, 463
M R C Biochemical and Clinical Magnetic Research Unit, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom.
K. van Dam, 1
E. C. Slater Institute for Biochemicul Research. University of Amsterdam, Plantage Muidergracht 12, 1018 T V Amsterdam, The Netherlands.
H. Weiss, 199
Institut fur Biochemie, Heinrich- Heine- Universitat DusseldorJ; UniversitatsstraJe I , W-4000
Dusseldorf I, Germany.
H.V. Westerhoff, 1
E. C. Slater Institute for Biochemical Research, University of Amsterdam, Plantuge Muidergracht 12, 1018 T V Amsterdam, The Netherland~s.
M. Wikstrom, 217
Helsinski Bioenergetics Group, Department of Medical Chemistry, University of Helsinski,

Siltavuorenpenger 10, SF-001 70 Helsinski, Finland.
M. Yamaguchi, 265
Division of Biochemistry, Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, 10686 North Torrey Pine5 Road, La Jolla, CA 92037, U S. A.


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xvii

Contents
Introduction, by L. Ernster . . . . .
Some reviews on topics related to the history
List of Contributors . . . . . . .
Contents . . . . . . . . . .
Non-conventional abbreviations . . . .

. . . . . . . . . . . . . v
ofbioenergetics
. . . . . . . . xi
...
. . . . . . . . . . . . .
~111
. . . . . . . . . . . . .
xvii
. . . . . . . . . . . . . xix

1. Thermodynamics and the regulation of cell functions
H. V. Westerhoffand K. van Dam . . . . . . . . . . . . . . . I
2. Chemiosmotic systems and the basic principles of cell energetics

V.P. Skulachev . . . . . . . . . . . . . . . . . . . .
31
3. Bacteriorhodopsin
15
R. Renthal . . . . . . . . . . . . . . . . . . . . .
4. High-resolution crystal structures of bacterial photosynthetic reaction centers
J. Deisenhofer and H. Michel . . . . . . . . . . . . . . . . 103
5. The two photosystems of oxygenic photosynthesis
B. Andersson and L. -G. FranzPn . . . . . . . . . . . . . . . 121
6. NADH -ubiquinone oxidoreductasr
T.P. Singer and R.R. Ramsay . . . . . . . . . . . . . . . . 145
7. Progress in succinate: quinone oxidoreductase research
L. Hederstedt and T. Ohnishi . . . . . . . . . . . . . . . . 163
8. Mitochondrial ubiquinol-cytochrome c oxidoreductase
G. Bechmann, U. Schulte and H. Weiss . . . . . . . . . . . . . 199
9. Cytochrome oxidase: notes on structure and mechanism
T. Haltia and M . Wikstrom . . . . . . . . . . . . . . . .211
10. Cytochrome c oxidase: tissue-specijic expression of isoforms and regulation of activity
B. Kadenbach and A. Reimann . . . . . . . . . . . . . . . . 241
I I . The energy-transducing nicotinamide nucleotide transhydrogenase
Y. Hatefi and M. Yamaguchi . . . . . . . . . . . . . . . . 265
12. The structure and assembly of A T P synthase
G.B. Cox, R.J. Devenish, F. Gibson, S . M . Howitt and P. Nagley . . . . . . 283
13. The reaction mechanism of F,,F,-ATP synthases
R.L. Cross . . . . . . . . . . . . . . . . . . . . .
311
14. Inorganic pyrophosphate and inorganic pyrophosphatases
M . Baltscheffsky and H. Baltscheffsky
. . . . . . . . . . . . . 331
15. Mitochondrial calcium transport

349
C. Richter . . . . . . . . . . . . . . . . . . . . .
14. Metabolite carriers in mitochondria
F. Palmieri and R. Kramer . . . . . . . . . . . . . . . . . 359
17. The uncoupling protein thermogenin and mitochondrial thermogenesis
J. Nedergaard and B. Cannon . . . . . . . . . . . . . . . . 385
18. Hormonal regulation of cellular energy metabolism
J.B. Hoek
. . . . . . . . . . . . . . . . . . . . .
421
19. The study of bioenergetics in vivo using nuclear magnetic resonance
G.K. Radda and D.J. Taylor . . . . . . . . . . . . . . . . 463
20. Recent advances on mitochondrial hiogenesis
A. Chomyn and G. Attardi . . . . . . . . . . . . . . . . . 483

Index.

.

.

.

.

.

.

.

.

.

.

.

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.

.

.

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.

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511


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xix

Non-conventional abbrevations
ETP
electron-transport particles
ADP/ATP carrier (adenine
EXAFS
extended X-ray absorption fine
nucleotide translocator)
structure
AcPyAD 3-acetylpyridine adenine dinuclefructose- 1,6-biphosphatase
FBPase
otide
carbon ylcyanide p-fluoroAcPyADP 3-acetylpyridine adenine dinucle- FCCP
methoxyphenylhydrazone
otide phosphate
ferredoxin
Fd
a-aminoisobutyrate
AIB
soluble subcomplex of quinolFRD

adenosine-5’-phosphosulphate
APS
fumarate reductase
BChl
bacteriochlorophyll
FSBA
p-sulfonyl benzyl-5‘-adenosine
brown fat mitochondria
BFM
follicle stimulating hormone
BHM
FSH
beef heart mitochondria
HA
BLM
hydroxyapatite
beef liver mitochondria
high mobility group
BPh
HMG
bacteriopheophytin
bR
HMG-COA hydroxymethyl-glutaryl-coenbacteriorhodopsin
bovine serum albumin
zyme A
BSA
7-(n-heptadecyl)mercapto-6-hyBzATP
3’-0-(4-benzoyl)benzoyl
droxy-5,8-quinolinequinone
adenosine 5‘-triphosphate

cyclic adenosine monophosphate HOQNO 2-n-heptyl- 1,4-hydroxyquinocAMP
carbonylcyanide p-chlorophenline-N-oxide
CCCP
HQNO
hydroxyquinoline-N-oxide
ylhydrazone
hR
halorhodopsin
coenzyme Q (ubiquinone)
COQ
heat shock protein
cytochrome oxidase (cytochrome hSP
cox
IDH
isocitrate dehydrogenase
c oxidase)
IMAC
inner membrane anion carrier
cAMP receptor protein
CRP
insulin-degrading enzyme (insuliINS
cytochrome
CYt
nase)
p-diazobenzene sulfonate
DABS
DAN-ATP 1,5-dimethylaminonaphtoyl
Ins-1,4,5-P3 inositol-l,4,5-triphosphate
3‘-o-ATP
a-ketoglutarate dehydrogenase

KGDH
N ,N’-dimethyl-dodecylamine-NN,N‘-dicyclohexylcarbodiimide
LDAO
DCCD
oxide
dichloromethylurea (diurone)
DCMU
linear electric field effect
LEFE
DEA
diethylammonium acetate
DMBIB
light-harvesting complex I
2,5-dibromo-3-methyl-6-isopro- LHCI
MCD
pylbenzoquinone
mutagenic circular dichroism
MIGB
m-iodobenzylguanidine
DMSO
dimethylsulfoxide
mosaic non-equilibrium thermoDTNB
5,5’-dithio-bis-(2-nitrobenzoate) MNET
dynamics
EDTA
ethylenediamine tetraacetate
EEDQ
p-methoxyacrylates
MOA
N-(ethoxycarbonyl)-2-ethoxymatrix protease

MP
1,2-dihydroquinoIine
matrix processing peptidase
EGTA
ethylene-bis(oxyethylenenitri1o)- MPP
MPP’
N-methyl-4-phenylpyridinium
tetraacetate
ENDOR
N-methyl-4-phenyl-tetrahydroelectron nuclear double reso- MPTP
pyridine
nance
ER
m-TERF mitochondrial termination factor
endoplasmic reticulum
ETHI-57 N,N‘-dibenzyl-N,N’-diphenyl- m-TF
mitochondrial transcription factor
1,2-phenylene-diacetamide
AAC


xx
mTF
MVC
NA
Nbf-C1
NEM
NET
OGC
ORF

OSCP
PBF
PC
PCr
PDE
PDH
PEP
PEP
PFK
PHM
PIC
PM
PME
pp,
PPase
PQ
prot K
PSI
PSI1

mitochondria1 transcription factor
maximal volume contraction
nicotinamide
4-chloro-7-nitrobenzofurazan
N-ethylmaleimide
non-equilibrium thermodynamics
oxoglutarate carrier
open reading frame
oligomycin sensitivity conferring
protein

presquence binding factor
plastocyanine
phosphocreatine
phosphodiesters
pyruvate dehydrogenase
phosphoenolp yruvate
processing-enhancing protein
phosphofructokinase
pig heart mitochondria
phosphate carrier
purple membrane
phosphomonoesterase
inorganic pyrophosphate
inorganic pyrophosphatase
plastoquinone
proteinase K
photosystem I
photosystem I1

propylthiouracil
coenzyme Q (ubiquinone)
Q
quinol-fumarate reductase
QFR
RC
reaction center
RCR
respiratory control ratio
RLM
rat liver mitochondria

RR
resonance Raman
S-13
5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide
SDH
succinate dehydrogenase (soluble subcomplex of SQR)
SMP
submitochondrial particles
succinate-quinone reductase
SQR
(Complex 11)
sarcoplasmic reticulum
SR
sensory rhodopsin
SR
tbh
tert-butylhydroperoxide
TCA cycle tricarboxylic acid cycle
tetrameth yl-p-phenylenediamine
TMPD
TNBS
2,4,6-trinitrobenzene sulfonate
TNM
tetranitromethane
TPBtetraphenylboron anion
TTFA
2-thenoyltrifluoroacetone
TUTase
terminal uridyl transferase
UCP

uncoupling protein
UHDBT S-n-undecyl-6-hydroxy-4,7-dioxobenzothiazol
UHNQ
3-n-undecyl-2-hydroxy1 ,4-naphthoquinone

PTU


L. Ernster (Ed.) Molecular Mechanisms in Bioenergerics
0 1992 Elsevier Science Publishers B.V. All rights reserved

1
CHAPTER 1

Thermodynamics and the regulation of
cell functions
HANS V. WESTERHOFF'%2
and KAREL van DAM]
' E .C. Slater Institutejor Biochemical Research, University of Amsterdam,
Plantage Muidergracht 12, NL-I018 TV Amsterdam, The Netherlands and
2Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121,
NL-1066 C X Amsterdam, The Netherlands

Contents
1. Principles
1.I. Free-energy transduction is essential for life
1.2. The energetics do not necessarily control cell function
1.3. Kinetic versus thermodynamic (energetic) control
1.4. Linearity and control
1.5. Precise analyses of control

1.6. Thermodynamic control analysis
1.7. Hierarchies in the control of energy metabolism
1.8. Signal transduction
1.9. Mosaic non-equilibrium thermodynamics and the central role of
free energies in control and regulation
2. Control of free-energy metabolism
2.1. Control analyses
2.2. Is the control kinetic or thermodynamic'?
2.3. Is ApHthe energetic intermediate?
3. Energetics of microbial growth
3. I . Description of growth by MNET
3.2. Control of microbial metabolism
3.2.1. Control by substrates
3.2.2. Control by enzymes
3.2.3. Control by intermediates?
3.2.4. Control by free energy
3.2.5. Control of metabolism by energetics; the yeast case
3.2.5.1. Glycolysis
3.2.5.2. Role of mitochondria
4. Energetics and the control of gene expression
4.1. The intracellular need for ATP
4.2. Do the energetic properties of cells ever change?
4.3. Does gene expression change when the energetics change?
5. Concluding remarks

2
2
3
4
6

7
8
9
14
14
17
17
18
19
19
23
24
24
25
26
26
21
21
28
28
28
30
30
31


L

Acknowledgement
Symbols

31
31

References

32

1. Principles
1.1. Free-energy transduction is essential for life
The paradox noted by Schrodinger ([l], see also ref. [ 2 ] )that whilst the universe progresses
towards maximum chaos, living systems seem to d o the opposite, illustrates the special importance biology has for thermodynamics and vice versa. Because living systems tend to operate
in isothermal, isobaric conditions more than in conditions of heat isolation and constant volume, the same paradox is more properly formulated as the question why living systems seem
to increase in free energy, whilst experience and thermodynamics tell us that processes tend
towards minimal free energy [2,3]. Free energy here represents the Gibbs free energy G (=
U+p V - T S ) . The tendency of G towards a decrease comprises the tendency of the true energy
U towards a decrease, the tendency of volume V to decrease and the tendency of entropy S to
increase.
The resolution of Schrodinger’s paradox has many aspects, the ultimate of which is one of
the fundamental problems of biology. The first aspect is that many living systems operate at
steady state and therefore d o not increase in free-energy content. This point does not quite
resolve the problem, however, because an essential aspect of living systems is that processes
occur in them. For processes to occur with net effect, they need to be driven by a free-energy
difference. Consequently, the occurrence of processes is necessarily accompanied by the destruction (dissipation) of free energy (production of entropy). Just to maintain the steady-state
processes in living systems, free energy must be dissipated. Living systems are able to destroy
free energy without decreasing in free-energy content due to their ability to import free energy
[4]. Often, this free energy has the form of the import of chemical compounds that are richer in
free energy than the products that are excreted.
When looking at a living system as a thermodynamic black box, this import of free energy
solves the paradox. However, inspection of the molecular contents of a living system reveals

another paradox. Like ‘dead’ systems, living systems house many processes that run downhill
in the thermodynamic sense; the free-energy content of their substrates exceeds that of their
products, hence they dissipate free energy. Here, enzymes serve to enhance the rate of a process
that would otherwise also occur, be it at a much slower a rate. What is more characteristic of
(though not unique to) living systems is that they also house reactions that run uphill in the
thermodynamic sense, i.e. reactions in which free energy is increased. Examples are the synthesis of complex molecules or new cells, the synthesis of ATP for use in muscle contraction and
the uptake of food from the cellular environment.
Special biological machines are needed to accomplish such tasks. They d o this by coupling
the reaction that is thermodynamically uphill to a reaction that is thermodynamically downhill,
such that the overall process always dissipates free energy whilst free energy is being stored in
the uphill process. These biological machines may be proteins, or entire metabolic networks.
These biological free-energy transducers can only support the uphill reaction when their
inner workings are in good order, i.e., when they d o not ‘slip’ too much, and when the downhill


3

reaction that is driving them has sufficient input free energy. Clearly, input free energy and the
free-energy transducing machinery are essential for the function of living systems.

1.2. The energetics do not necessurily control cell junction
The fact that something is essential for some function does not imply that it also controls or is
even involved in the regulation of the latter. Indeed, a free-energy transducer may have excess
input free-energy available to it. An example is that of a proton pumping ATPase in the
presence of 10 mM ATP, whilst the K , of the enzyme is 0.1 mM. On the other hand, under
conditions of extreme starvation, or in the presence of agents that compromise the mechanism
of the free-energy transducer, the free-energy transduction must become ‘limiting’. It is clear
that the question to what extent the energetics control and perhaps even regulate cellular
functions is legitimate and should allow for an answer that depends on the conditions under
which the cell operates. What then would be a sensible definition?

It seems fairly straightforward to translate the question whether a given free energy, or a
given free-energy transducer controls a function, say a flux J , into: What is the effect on J if I
modulate the magnitude of the free energy, or the activity of the transducer, respectively?
However, this question is not precise enough, because its answer depends on the magnitude of
the modulation. When the modulation is taken as a complete knock out, the question really
asks for what is essential for the cellular function. As indicated above, the answer will then be
that both the input free-energy and the transducer are essential.
The knock-out definition and particularly the parallel experiment is nonphysiological in that
the information it yields derives in part from a condition that is very far removed from the
physiological condition of the cell. It seems more natural to modulate the activity of the
transducer, or the magnitude of the free energy more subtly and then measure the effect on flux
J . The effect on J relative to the magnitude of the modulation of the free energy or the
transducer may then be taken as a measure of the control exerted by either of the latter on the
flux. When the modulation is made small, this measure of the control becomes independent of
the magnitude of the modulation. To detach this measure of control of the dimensions (units)
of the two compared properties, the modulation and the change in flux may be expressed in
relative terms. Consequently, a coefficient serving to quantify the control of a transducer o n a
flux J m a y be defined as the percentage change in the flux J resulting from a 1% increase in the
activity of the transducer.
When one substitutes p% for the I % in the above definition and takes the limit to infinitely
small p , then the above definition becomes a differential, which brings the bonus that it can
become the subject of mathematical analyses. It is this definition that is called the control
coefficient in Metabolic Control Analysis (MCA) [5,3,6].This refinement need not bother us at
this moment, however.
What is more important a t this point is that, with this definition of control, one has a way to
establish unequivocally to what extent the free energy or a transducer controls a flux or any
other functional property of a living system. This extent (e.g., the percentage change in J) may
turn out to be very close to zero, in which cqse one has a rationale to state that that particular
free energy or transducer does not control the functional property of interest under the condition of interest, even though it is beyond doubt that the free energy and the transducer are both
essential for cell function. Of course, if that percentage change (i.e., the control coefficient) is

close to one, then the functional property is proportional to the activity of the free-energy
transducer, so that it is reasonable to summarize the situation by stating that the transducer
exerts full control on the functional property.


Interestingly, this definition of control allows for subtlety in the analysis of control of cell
physiology that has turned out to be important for the understanding thereof. For instance, a
control coefficient may turn out to be 0.5, implying that a 1 % change in the transducer affects
the functional property by 0.5%. Without this subtlety, one would have had to conclude that
the transducer is without control or fully in control of the functional property, since more
subtle categories would not exist. Also, it allows the scientific analysis of the intuitive notion
that the control of a cellular function may be partly in one factor, partly in a second factor and
for the rest in a third factor. The definition of control coefficients provides for a method to
demonstrate that three factors are indeed important in such a case and what their relative
importance is.
Let us suppose that for a particular function, say growth rate, a free-energy transducer has
a control coefficient of 0.9. The transducer may then be said to be virtually in control of growth
(‘growth limiting’). Is this sufficient reason to conclude that the transducer is also involved in
the regulation of growth? The answer to this question is no. One speaks of regulation when
growth rate is changed as a response to a certain change in environment [7,8] (or [9] when it is
buffered against such environmental changes). Such regulation may occur without involving a
change in the activity of the transducer; it may proceed through activation of a different
reaction in the system, which has perhaps a control coefficient of only 0.1 with respect to
growth rate, but is activated to a large extent. To quantify regulation one refers to the magnitude and effects of actual modulations that occur in a system that is being regulated. Control
coefficients refer to the fact whether an experimental modulation of a reaction would affect the
functional property of interest without asking whether or not such a modulation occurs in a
physiological transition.

1.3. Kinetic versus thermodynamic (energetic) control
In this section on principles, it may be appropriate to discuss the distinction that has been made

in the literature [e.g. 10,l I] between thermodynamic and kinetic control. If one discusses the
control of a concentration, there is a relatively straightforward definition of thermodynamic
control of a concentration [XI by another substance S: [XI is controlled thermodynamically by
S if the reaction converting S to X is and remains at equilibrium. In this case, [XI equals the
equilibrium constant times the concentration of S.
However, often the term thermodynamic control is used with respect to a flux; this pertains
to a non-equilibrium situation. Let us consider a conversion of a substrate S to a product P,
which has a free-energy difference AG = ,us- p p as its driving force. ,us and puprefer to the
chemical potentials of S and P, respectively (,us= p ! + RTln[S]). At equilibrium, the rate of the
process is solely determined by the driving force; independent of any parameter value, whenever the driving force is zero, the flux is zero; purely thermodynamic control therefore, since the
driving force is a thermodynamic property. Away from equilibrium, however, the reaction rate
may be determined independently by both [S] and [PI, hence not by the driving force alone. The
special case that the reaction rate is determined by the driving force alone, may sensibly be
called the case of ‘thermodynamic control’.
As an aside, we may mention that thermodynamic control is somewhat of a misnomer. For,
concentration and chemical potential of a substance are related by a simple exponential function. And, more strictly speaking, solution kinetics tends to use activities rather than concentrations in its rate equations, where activities are purely thermodynamic properties. Indeed the
rate of conversion of S to P can be written as a function of the chemical potentials of S and P
13,121.