BIOENERGETICS


New Comprehensive Biochemistry

Volume 9

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Utrecht

ELSEVIER
AMSTERDAM. NEW YORK . OXFORD


BIOENERGETICS

Editor

L. ERNSTER
Stockholm

1984

ELSEVIER
A M S T E R D A M . NEW YORK . OXFORD

ZJ 1984 Elsevier Science Publishers B.V.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Bioenergetics
(New comprehensive biochemistry: v. 9)
Includes bibliographies and index.

1. Bioenergetics. 2. Energy metabolism.
1. Ernster, L. 11. Series.
QD415.N48 vol. 9 574.19’2 s [574.19’283] 84-21273
[QHSlOI
ISBN 0-444-80579-6

Printed in The Netherlands


Introduction

“Research is to see what ecervbod). has seeti utid think uhat nohod, has 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, is
also the main topic of the present volume. This trend is, however, relatively new; for
example, it was not reflected in the contents of the previous volume on Bioenergetics
in this series that appeared in 1967. As an introduction to the chapters that follow it
appears appropriate, therefore, to give a brief historical background of these new
developments. 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 IngenHousz, 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 uitalism, 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


VI

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, i t became increasingly evident that living organisms
could produce 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 on

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 uitale that was provided by, and dependent on, an 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 Buchner, 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 Buchner’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 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.



v11
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 r6le is to enhance the activity of the
iron-containing respiratory enzyme, Atmungsferment. Shortly 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 to the
‘charcoal model’ and Wieland to the ‘platinum model’ in attempting to 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 at one end and Atmungsferment at the other, thereby bridging the
gap in opinion between Warburg and Wieland. Using a particulate preparation from
mammalian heart muscle, Keilin and Hartree subsequently showed Warburg’s
Atmungsferment to be identical to Keilin’s cytochrome u 3 . 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/O 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 ,f3-hydroxybutyrate or reduced nicotinamide adenine dinucleotide as substrate.


VlII

Although mitochondria had been observed by cytologists since the 1840s. the
elucidation of their function had to 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 ATP-synthesizing system.
In the following years research in many laboratories was focused 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 non-phosphorylated high-energy compounds
as intermediates between electron transport and ATP synthesis, a chemical coupling
mechanism envisaged by several laboratories and first formulated in general terms
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


IX

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 from 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, 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 intect 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 thinlung 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. I t
was shown that electron-transport 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 transportlinked 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 energylinked nicotinamide nucleotide transhydrogenase, the synthesis of inorganic pyrophosphate, the active transport of ions and metabolites, mitochondria1 thermogenesis in brown adipose tissue and light-driven ATP synthesis and ion transport in
Halobacteria. The chapters of this volume give an overview of our present state of
knowledge concerning these processes.
The central problem in this field at present is to clarify the mechanisms involved
in membrane-associated energy transduction at the molecular level. What are the



X

molecular mechanisms by which energy-transducing catalysts translocate protons
across the membrane? Is the generation of a proton gradient the primary event in
energy conservation or is it preceded by chemical, e.g., conformational changes in
the catalysts involved? Is communication by way of a transmembrane proton
gradient the only means by which energy-transducing catalysts interact or are there
mechanisms for more direct, ‘localized’ interactions between them within the membrane? How is the biosynthesis of energy-transducing catalysts regulated, e g , in
relation to subunit stoichiometry or, in the case of eukaryotes, to the coordination of
nuclear and organellar gene expression? How are the subunits of energy-transducing
catalysts processed and assembled in the membrane, and what is the relationship of
these processes to the energy-state of the cell?
These are some of the current problems that are discussed in various chapters of
this volume. Progress regarding these problems has long been dependent on knowledge of the structures, reaction mechanisms and biogenesis of the individual energytransducing catalysts and their relationship to, and interactions within, the membranes in which they reside. Such information has been forthcoming during the last
few years at an accelerating rate and further rapid progress can be foreseen. Looking
at the field as a whole, one is left with the impression that, perhaps for the first time,
bioenergeticists are taking full advantage of the powerful arsenal of methods and
concepts of molecular biology and, vice versa, molecular biologists are becoming
genuinely engaged in the fundamental problems of bioenergetics. What we may be
witnessing is the fall of the last bastion of vitalism, the transition of membrane
bioenergetics into moleculur bioenergetics.
In 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 made it possible to publish this volume while its contents are still reasonably
up-to-date. I also wish to thank my colleague Kerstin Nordenbrand at the Arrhenius
Laboratory for her valuable help with the editorial work, and Jim Orr, Desk Editor
of the Biomedical Division. Elsevier Science Publishers B.V., for friendly and

efficient cooperation.
Lars Ernster
Department of Biochemistry
Arrhenius Laboratory
University of Stockholm
S-106 91 Stockholm
Sweden


Some reviews on topics related to the history
of bioenergetics
( I n chronological order of appearance)

Rabinowitch. E.I. (1945) Photosynthesis and Related Processes. Interscience, New York.
Lindberg, 0. and Ernster, L. (1954) Chemistry and Physiology of Mitochondria and Microsomes.
Springer-Verlag. 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 (Brachet. J. and Mirsky, A.E., eds.)
Academic Press, New York, Vol. 11, pp. 299-421.
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.
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, Springer-Verlag,
Heidelberg, Vol. 5 , pp. 7-56.
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.
(Skulachev. V.P. and Hinkle, P.C., eds.) pp. 3-46. Addison-Wesley, Reading, MA.
Slater, E.C. (1981) A short history of the biochemistry of mitochondria. In Mitochondria and Microsomes. (Lee, C.P., Schatz, G . and Dallner, G . , eds.) pp. 15-43. Addison-Wesley, Reading, MA.
Tzagoloff, A. (1982) Mitochondria. Plenum Press, New York.
Hoober, J.K. (1984) Chloroplasts. Plenum Press. New York.


Non-conventional abbreviations
AcPyAD
AMP- PN P
ATPS ( fl and/or y )
BAL
CAT
CCCP (CICCP)
DBMIB
DCCD
DCMU
DMPC
DMSO
DNP
DNP-INT
DTNB

EDTA
EGTA
ELISA
ETF
FCCP
FPLC
FSBA
HMHQQ
HOQNO
NBDCl
NEM
OSCP
PMS
PP,
PPase

Q
RuBP
SRP
TPP+
UHDBT
1799

acetyl pyridine adenine dinucleotide
adenyl imidodiphosphate
thiophosphate analogs of ATP
2.3-dimercaptopropanol (British Anti Lewisite)
carboxyatractyloside (carboxyatractylate)
carbonylcyanide p-chloromethoxyphenylhydrazone
2,5-dibromo-3-methyl-6-isopropylbenzoquinone

N , N’-dicyclohexylcarbodiimide
3-(3,4-dichlorophenyl)-l -dimethylurea
dimyristoyl phosphatidylcholine
dimethylsulfoxide
2,4-dini trophenol
2-iodo-6-isopropyl-3-methyl-2,4,4’-trini
tro-diphenylether
5,5’-dithiobis( 2-nitro-benzoate)
ethylenediamine tetraacetate
ethyleneglycol tetraacetate
enzyme-linked immunosorbent assay
electron-transferring flavoprotein
carbonylcyanide p-fluoromethoxyphenylhydrazone
fast protein liquid chromatography
p-fluorosulfonylbenzoyl-5-adenosine
7-( n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone
2-n-heptyl-4-hydroxyquinoline N-oxide
4-chloro-7-ni tro-2-oxal-l,3-diazole
N-ethylmaleimide
oligomycin sensitivity conferring protein
phenazine methosulfate
inorga.nic pyrophosphate
pyrophosphatase
ubiquinone

ribulose-l,5-biphosphate
signal recognition particle
triphenylphosphonium ion
5-n-undecyl-6-hydroxy-4,7-dioxobenzo
thiazol

2,6-dihydroxy-l, 1,1,7,7,7-hexafluoro-2,6-bis-(
trifluoromethy1)hep tanone-4-[ bis(hexafluoroacetyl)acetone]


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

V
XI
XI1

Chapter 1. Thermodynamic aspects of bioenergetics. by K . Van Dam and
H . V. Westerhoff

1

1. Introduction . . . . . . . . . . . . . .
2 . Simple thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . (Thermo-)hnetics . . . .
3.1. Theprinciple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The physical constraint [S] [PI constant
..........
3.3. Short notation for the therm
4 . A mosaic in non-equilibrium thermodynamics (M
4.1. Facilitated flux across a membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Coupling between diffusion fluxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Coupling between chemical reaction and flux . . . . . . .
.....................

4.4. Leaks and slips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Application of MNET to biological free-energy converters . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Bacteriorhodopsin liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Oxidative phosphorylation in mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
....
..
5.2.1. Stoicheiometries . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2. Localization of the high free-energy proton . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.3. Slipping proton pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Bacterial growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
2
4
4
6
10
11
12
12
13
13
15
15
18
18
21
21

23
25
26

Chapter 2. Mechanisms of energy transduction. by D . Nicholls

29

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . The basic features of the chemiosmotic theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Energy flow pathways in mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. The four postulates for the experimental verification of the chemiosmotic theory
2.3.1. The energy-transducing membrane is topologically closed and has a low proton
permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29
29
29
30
31

+

31


XIV

2.3.2. There are proton- (or OH-)-linked solute systems for metabolite transport and

osmotic stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.3. The ATP synthase is a reversible proton-translocating ATPase . . . . . . . . . . . . . . .
2.3.4. The respiratory and photosynthetic electron-transfer pathways are proton pumps
operating with the same polarity as does the ATP synthase when hydrolyzing ATP .
3. The proton circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. The potential term - proton electrochemical potential . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. Membrane potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Intrinsic indicators of membrane potential . .
...................
3.1.3. p H gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Proton conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. The special case of brown fat mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Respiratory control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Reversed electron transfer and the proton circuit driven by ATP hydrolysis . . . . . . . . . .
4 . Coupling of the proton circuit to the transport of divalent cations . . . . . . . . . . . . . . . . . . . . .
5 . Is the proton circuit in equilibrium with the bulk aqueous phases on either side of the
membrane? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Kinetic evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Thermodynamic anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32
32
33
34
35
35
37
37

38
38
39
39
41
43
46
47
47
47

Chapter 3. The mitochondria1 respiratory chain. by M . Wikstrom and M . Saruste

49

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . General survey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. The central dogma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Thermodynamic limits for mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Occupancy and mobility of the respiratory chain in the membrane
..........
..........
2.4. Functional domains in the membrane . . . . . . . . . . . . . . . . . . . .
3. Cytochrome oxidase or complex IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Topography and image reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Catalytic activity . .
3.4. Interaction with cyto
3.5. Mechanism of electron transfer and reduction of 0, . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. The redox centres and their location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.7. Energy conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.1. On the mechanism of proton/electron annihilation . . . . . . . . . . . . . . . . . . . . . . .
3.7.2. On the mechanism of proton translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.3. Role of subunit I11 in proton translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . The cytochrome bc, complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Composition and structure .
......
4.2. Cytochrome b . . . . . . . . . .
4.3. Cytochrome c, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. The Rieske FeS protein . . . . . .
4.5. Subcomplexes and image reconstruction of membrane crystals . . . . . . . . . . . . . . . . . . .
4.6. Topography of redox centres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49
51
51
52
54
57
57
57
59
59
59
60
62
64
65
66
67

69
69
72
73
74


xv
U biquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.1. Redox properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.2. Ubiquinone in the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8. Pathway of electron transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.9. Proton translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.10. Reconstitution of Complex I11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. The NADH-ubiquinone reductase complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Structure . . . . . . . . . . . . . . . . . . . . .
.............................
5.2. Iron-sulphur centres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Inhibitors and electron transfer pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Energy conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . .
.................................................

74
74
76
76
80
81

81
82
83
85
85
86
87

Chapter 4. Photosynthetic electrori transfer. by B.A. Melundri und G. Venturoli

95

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Reaction centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. General remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Experimental approaches to the study of reaction centers
3. The reaction centers of photosynthetic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Composition and protein structure
................
3.2. D, the bacteriochlorophyll dimer a
3.3. A, bacteriopheophytin as an intermediate electron acceptor . . . . . . . . . . . . . . . . . . . . .
3.4. A , quinone as a primary electron acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. A, quinone as secondary acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Photosystem I of higher plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Polypeptide and pigment composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Dl. a chlorophyll u dimer as electron donor . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Al., chlorophyll u as intermediate acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. A [ .2 . the electron acceptor X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. A 3 . iron sulphur centers as secondary acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Photosystem I1 of higher plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1. Polypeptide and pigment composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Dl1., chlorophyll a as primary electron donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. A l l . pheophytin u as intermediate electron acceptor
5.4. All., plastoquinone as primary electron acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. A,l.y plastoquinone as tertiary electron acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6. Dll., the secondary donor to PSI1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. The cytochrome b / c , complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. General remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Isolation procedures and properties of the complexes . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1. The ubiquinol-cytochrome c oxidoreductase of photosynthetic bacteria . . . .
6.2.2. The b, //complex of higher plant chloroplasts and cyanobacteria . . . . . . . . . . . . .
6.3. Cytochromes of b type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Cytochromes of c type
6.5. The high-potential Fe-S
6.6. The mechanism of electron transfer within the b / c , complex . . . . . . . . . . . . . . . . . . . . .

95
96
96
98
99
99
100
103
103
104
105
105
106
107

107
107
111
111
111
112
115
116
116
117
117
118
118
118
119
120
121
122

4.7.

..
..

.

.
.
.



XVI
7. Oxygen-evolving complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. General remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Involvement of manganese and other cofactors
.........................
7.3. Kinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Cytochrome b-559 .
..................................................
9. The redox interaction between complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.1. The secondary electron donors to bacterial and PSI reaction centers . . . . . . . . . . . . . . .
9.2. The role of quinones in the interaction between complexes . . . . . . . . . . . . . . . . . . . . . .
9.3. The reduction of NADPf by photosystem I . .
............
10.Membrane topology and proton translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125
125
125
127
131
132
132
133
135
136
142

Chapter 5. Proton mofive A T P synthesis. by Y. Kuguwu


149

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Structure of H + ATPase (FoF, ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Subunits of FoFl and its reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Subunits of F, and F,, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Organization of subunits in F,, F, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Primary structure and gene analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. FoFl gene . . . . . . . . .
..................
2.2.2. Homologies in primary
.........................
2.2.3. Chemical modification of the primary structure . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Secondary structure and the subunits of FoFl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Tertiary and quaternary structure of FoFl .
2.4.1. Stepwise reconstitution of FoFl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.2. Crystallographic analysis of F, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.................
2.4.3. Dynamic conformational change of F, and F( . . . . .
.................
3. Function of FoFl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Phosphorylation in biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. ATPase and H + transport in intact membranes . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Electrochemical potential of H + localized and delocalized . . . . . . . . . . . . . . . . .
3.1.3. H + / A T P r a t i o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. FoFl-Proteoliposornes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. ATP synthesis driven by A p H + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1. Ion gradient applied to FoF, proteoliposornes . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2. Electric field applied to FoF, proteoliposornes . . . . . . . . . . . .

3.4. Formation of Fl-bound ATP without A D H + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Mechanism of the H + ATPase reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Stereochemistry of the ATPase reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1. Stereochemical course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2. Cation-dependent diastereoisomer preference . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Energetics of the F,-bound nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1. Binding sites of nucleotides and inlubitors in F, . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2. Energy requiring step in ATP synthesis in FoFl . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Uni-site and multi-site kinetics of F, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1. Positive cooperativity in V,,,,, and negative cooperativity in K , . . . . . . . . . . . . . .
4.3.2. Control of ATPase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

149
150
150
150
151
152
152
153
155
156
158
159
160
160
160
160

161
162
163
164
164
166
167
167
167
168
170
170
171
172
172
174


XVlI
4.4. Coupling of proton flux and ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.1. Mechanism of proton translocation . . . .
...
....
4.4.2. Release of ATP from F,F,. ATP by Ap, + . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.3. A new model: acid-base cluster hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174
174

175
176
179
180

Chapter 6. The synthesis and utilization of pyrophosphate. by M . Baltscheffsky
and P . Nyren

187

1.
2.
3.
4.
5.
6.

Introduction . . . . . .
.................................................
Properties of inorganic
ophosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Formation of inorganic pyrophosphate . . . . . . . .
Inorganic pyrophosphate as phosphate and energy
s ..............
Membrane bound pyrophosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The mitochondria1 membrane-bound PP, ase . . . . . . . . . . . . . . . . . . .
6.1. Electron transport-coupled synthesis of PP, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. PP,-synthesis in relation to ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Solubilization and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Resolution and reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 . The H + -PPiase from Rhodospirillurn rubrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Electron transport-coupled synthesis of PP, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. PP,-driven energy-requiring reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.1. PP,-induced changes in the redox state of cytochromes . . . . . . . . . . . . . . . . . . . .
7.2.2. PP,-induced carotenoid absorbance change . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.3. PP,-driven energy-linked transhydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.4. PP,-driven succinate-linked N A D f reduction . . . . . .
7.2.5. PP,-driven ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Mechanistic aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4. Solubilization and purification . . . . . . . . . . . . .
..........................
7.5. Reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 . Outlook . . . . . . . . .
..............................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

187
188
189
189
191
192
192
193
193
194
195
195
196
196

197
197
198
198
199
200
202
203
204

Chapter 7. Mitochondria1 nicotinamide nucleotide transhydrogenase. by J .
Rydstrom. B . Persson and H.-L. Tang
207
1 . Introduction
................................................
2 . Energy-linked transhydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Relationship to the energy-coupling system
..
............
2.2. Reaction mechanism and regulation . . . . .
..
............
2.3. Energy-coupling mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Properties of purified and reconstituted transhydrogenase from beef heart . . . . . . . . . . . . . . .
3.1. Purification and reconstitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Catalytic and regulatory properties . . . . . . . . . . . . . . . .
...
3.3. Proton translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


207
208
208
209
210
212
212
214
215
216


XVIll

Chapter 8. Metabolite transport in mammalian mitochondria. by K .F. LaNoue
and A . C. Schoolwerrh
221

1. Introduction
....................................
2. Identification of the transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..............................
rane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Molecular mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Kinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1. Proton co-transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.1. Glutamate transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.2. Pyruvate transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.3. Phosphate transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2. Neutral exchange carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1.2.1. a-Ketoglutarate/malate carrier . . . . .
5.1.2.2. Other electroneutral exchange transpor
.....................
5.1.3. The electrogenic carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3.1. Glutamate/aspartate carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.3.2. The adenine nucleotide carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Structural studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1. The adenine nucleotide carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2. The phosphate transporter . . . . . . . . . . . . . . . . .
..
5.2.3. Other transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . The influence of mitochondrial transporters on metabolic fluxes .
........
6.1. Overview and definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Control of respiration by the adenine nucleotide carrier . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Gluconeogenesis and the pyruvate transporter
....
..............
6.4. Ammonia formation by the kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.1. Acute regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.2. Chronic acidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221
223
225
227
228
229

231
232
233
234
235
235
236
236
236
238
241
241
246
247
247
241
250
254
256
257
259
261
262

Chupter 9. The uptake and release of calcium by mitochondria. by E . Carufoli and
G. Sottocasa

1.
2.
3.

4.

Earlyhstory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The 'limited loading' of mitochondria with C a 2 + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanism of the Ca2+ uptake process .
...........
..................
Molecular components of the calcium upta
5 . The reversibility of the Ca2+ influx system and the problem of a separate route for C a 2 + efflux
from mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . The Na+-activated Ca2+ release route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Calcium movements evoked by changes in the redox state of pyridine nucleotides
8 . Regulation of the mitochondrial Ca2+ transport process . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 . Mitochondria in the intracellular homeostasis of Ca2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . .
.............................................

269
271
273
274
271
278
281
282
284
286


XIX


Chapter 10. Thermogenic mitochondria, by J . Nedergaard and B. Cannon
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. The thermogenin concept
.................
2.1. The uncoupled state
2.2. The coupling effects of purine nucleotides . . . . . .
2.3. The high (but regulated) halide permeability . . . . . . . . . . . . . . . . . . . , . . . , , . . . , , . .
2.4. The matrix condensation during mitochondria1isolation . . . . . . . . . . . . . . . . . . . . . . . .
2.5. The existence of a purine nucleotide binding site on brown fat mitochondria. . . . . . , . , .
2.6. The ability of brown fat mitochondria to alter their capacity for heat production . . . . . . .
3. The manifestations and measurements of thermogenin
..............
3.1. GDP binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Immunoassays . . . . .
..........................................
3.4. GDP-sensitive permea
..........................................
4. The thermogenin molecule
....
................
5. The regulation of thermoge
........................................
5.1. Suggested non-free fatty acid mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Mediators secondary to free fatty acid release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.1. Free fatty acids . . . . . . . . . . . . . , . . . . . . . , . . . . . . . . . . . . , . . . , , , . . . . . . .
5.2.2. Acyl-CoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 . The regulation of thermogenin amounts . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , , . . . . , . . .
6.1. The expression of thermogenin

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

291
29 1
292
293
294
295
297
298
298
299
300
303
303
303
304
306
306
307
307
309
310
31 1
312

Chapter 11. Bacteriorhodopsin and related light-energy converters, by J . K. Lanyi 315
1. Introduction

...................................

...................................

2.1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . .
2.2. Chromophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Photochemical reactions . . . . . . . . . . . . . . . . . . . .
2.4. Proton transport .
.............................................
3. Halorhodopsin . . . . . . . . . . . . . . . . . . . . . .
................
3.1. Spectroscopic and molecular properties
................
3.2. Functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . .
4. Slowly cycling rhodopsin .
...............
5. Light-driven ion transport in the halobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31 5
318
318
323
325
331
333
333
335
337
331
341


Chapter 12. Biogenesis of energy-transducing systems, by N . Nelson and
H. Riezman
351
1. Introduction . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . .
2. Semiautonomous organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Organellar DNA . . . . . . . . . . . . , , . . , , . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . .
2.2. Organellar protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

351
352
352
354


xx
3. Import of proteins into chloroplasts. mitochondria and storage vesicles . . . . . . . . . . . . . . . . .
4 . Vectorial translation - biogenesis of secretory vesicles and acetylcholine receptor . . . . . . . . .
4.1. Biogenesis of chromaffin granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Biogenesis of the acetylcholine receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Vectorial processing - import of proteins into chloroplasts and mitochondria . . . . . . . . . . . .
5.1. Synthesis of cytoplasmic ribosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Binding of precursors to the organellar surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Transmembrane movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Processing of precursor and sorting into the correct compartment . . . . . . . . . . . . . . . . .
6 . Protein incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Assembly of functional protein complexes . . . . . . . . . . . . . . . . . . . . . .
8 . Regulation of membrane formation . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355

356
356
358
361
361
362
362
365
367
368
368
374

SubjectIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

379


1

Ernster (ed.) Bioenergefics
81 1984 Elseuier Science Publishers B. V.

CHAPTER 1

Thermodynamic aspects of bioenergetics
KAREL VAN DAM and HANS V. WESTERHOFF *
Laboratory of Biochemistry, B. C.P . Jansen Institute, University of Amsterdam,
Plantage Muidergracht 12, Amsterdam, The Netherlands


I . Introduction
By definition, bioenergetics is concerned with the transformations of energy in
biological systems. Thermodynamics, though originally focussing on how heat could
be used to do useful work in steam engines, was soon extended to analyze the
interconversion of many more forms of energy.
Yet, statements of classical thermodynamics are quantitative only when the
system studied is in equilibrium. Biology is interested in living systems. Such systems
are always out of equilibrium so that classical thermodynamics has a limited
potential for biology. The work of Onsager [I] suggested that thermodynamics could
be extended to the description of non-equilibrium systems. The theory behind the
application of this ‘near-equilibrium’ (21 non-equilibrium thermodynamics (NET) to
biological systems has been elaborated in great detail [3-51. It provided insight into
the thermodynamic implication of the coupling of (in terms of Gibbs free energy)
‘ uphill’ to ‘downhill’ processes and into the resulting thermodynamic efficiency of
energy coupling. Yet, this near-equilibrium NET never became as generally used,
and even accepted, as for instance the enzyme kinetics developed by Michaelis and
Menten. Reasons for this were the following.
( i ) The validity of the near-equilibrium non-equilibrium thermodynamics could
only be guaranteed if all processes were ‘close’ to equilibrium, where ‘close’ should
be interpreted as AG << 1.5 kJ/mol. Most of the interesting processes in living
systems are much farther from equilibrium.
( i i ) This near-equilibrium non-equilibrium thermodynamics described biological
systems as black boxes (some exceptions are found in Refs. 3, 7 and 8). I t lacked the
ambition of relating in- and output characteristics of the biological system to the
mechanisms of operation of the metabolism within that black box. It is precisely
those mechanisms that are of great interest to most biological chemists and physicists.
* Present address: National Institutes of Health, Building 2, Room 310, Bethesda, M D 20205. U.S.A.


2

( i i i ) On many points, near-equilibrium non-equilibrium thermodynamics seemed
to be in conflict with that was already known from enzyme kinetics: it predicted that
reaction rates would go to infinity when the substrate concentration would do so,
whereas enzyme catalyzed reactions exhibit a maximum rate.
After the extension of thermodynamics to near-equilibrium systems had thus
turned out to be of limited use in biological systems, ‘a number of authors
contributed to yet another extension of thermodynamics, i.e., to (biological) systems
in which most reactions are enzyme-catalyzed. The latter extension also allows one
to quantitatively relate the metabolic behaviour of biological systems to the characteristics of the enzymes within them. Meanwhile, this extension has been used to
extract mechanistic information from experimental data obtained in a number of
free-energy transducing systems.
Although it will burden the student of bioenergetics with some mathematical
gymnastics, we feel that further progress in the understanding of a number of, still
unsolved, elementary problems in bioenergetics, is impossible without quantitative
analysis of the functioning of biological free-energy transducing systems. Examples
of such problems are the extent of localization of the energy transducing protons;
the occurrence of ‘slip’ (see below) in the proton pumps; the stoicheiometries at
which protons are pumped; the extent to which specific enzymes control free-energy
transduction.
Therefore, we shall present here a taste of the modern thermodynamic approaches
to bioenergetics.

2. Simple thermodynamics
If thermodynamics would limit itself to the study of changes in the amount of energy
( U ) in a system, its application to biology would be rather dull. Energy ( U ) itself is
a ‘conserved’ quantity, i.e., it can neither be destroyed nor created. The interesting
part comes when it is realized that energy can appear in different forms which
generally have different capacities to do useful work. In turn, these capacities
depend on the type of system we are concerned with, e.g., heat has a large capacity
to do useful work (only) if there is a large temperature difference between different

parts of the system. Flux of a substance has a high capacity to do work, only if there
is a high difference in its chemical potential (i.e., concentration) between different
parts of the system. In the usual biological systems, there are no significant
temperature gradients, so that pure heat is not a very useful form of energy, at least
not in terms of doing work.
In an isothermal, isobaric system, the amount of energy that can be used to do
useful work is equal to the Gibbs Free energy, G (defined as U + PV - T S ) . Pure
heat in isothermal systems is a form of energy, the free-energy content of which is
zero. All spontaneous chemical and physical processes proceed in such a way that
some free energy is destroyed (‘dissipated’). It should be noted that this does not
imply that the free energy of a system has to decrease. When the system is in steady
state, its free energy is constant. The correct implication then is that more free


3

energy is imported into the system than is exported, such that the free energy of the
system plus its surroundings does decrease. The expenditure of free energy, as
fatalistic as it has been discussed at times, can also be seen from the more positive
side: free energy is the factor that makes processes run. For systems not too far from
equilibrium, it can even be shown that the rate of processes increases with the
amount of free energy spent in making them run. Moreover, such systems evolve in
such a way that the rate at which they dissipate free energy decreases with time [9]. It
is a simple consequence of this that evolution will stop, i.e., steady state will be
attained, when the free-energy dissipation cannot decrease any further: in the steady
state free-energy dissipation is minimal.
As stated above, free-energy (G) dissipation does not imply that energy ( U ) is
dissipated: it only implies that energy is (partly) transformed from free energy to
pure heat, which equals the product of temperature and entropy. Consequently, the
rate of free-energy dissipation is equal to the rate of entropy production multiplied

by the absolute temperature.
The so-called dissipation function (a) analyzes the rate of free energy dissipation
in terms of the different processes in which free energy is dissipated. If, for instance,
a chemical reaction occurs with a free-energy difference, AG,, and at a rate, Jchem,
whereas at the same time a substance S flows from a space in which it has a high
concentration to one where it has a low concentration, the rate at which the free
energy is dissipated is:

It may be noted that we define AG such that it equals the chemical potential of the
substrate minus the chemical potential of the product. We noted above that the
possibility of free-energy dissipation drives a reaction. Free-energy differences like
AG, and Aps in the above equation embody such a possibility: they act as forces that
drive the reaction. Other examples are: the contractile force on a muscle; the voltage
drop across an electrical resistance; the osmotic pressure on a semipermeable
membrane. The dissipation function consists of the sum of the products of fluxes
(currents) and the (thermodynamic) forces that drive them [4].
The dissipation function always has a positive value (according to the second law
of thermodynamics), but the sign of each of the separate flux-force couples is not a
priori defined. Thus, the negative contribution of a diffusion flux against a concentration gradient may be compensated by the positive contribution of a chemical
reaction proceeding at a high free-energy difference. This can, however, only occur if
the two processes are coupled in one way or another. Any independent (not coupled)
set of fluxes and associated forces must conform to the criterion of positive entropy
production [lo].
Generally speaking, a flux ( J ) in a system can depend on each of the forces ( ‘ X ’ )
in that system. Close to equilibrium it can be made feasible [I] that this function is
in general proportional, i.e.:

J , = L ; ,. XI

+ L,


.X,

+ . . . + L,,,. x,


4

The proportionality constant
is constant in the sense that it is independent of the
forces ( X ) .
The complete set of equations, relating fluxes and forces can be written in a
matrix form and is called the set of phenomenological equations. It was shown by
Onsager [l] that the matrix of the phenomenological proportionality constants is
symmetrical:

This reduces the number of independent constants in the system.

3. (Thermo-)kinetics
The phenomenological equations given above are limited in (demonstrated) validity
to near-equilibrium systems. They belong to the near-equilibrium NET approach
discussed in the introduction. They demonstrate some of the limitations of the
near-equilibrium approach in the sense that the relationship between the phenomenological constants ( L U )and the biochemical and biophysical mechanisms within
the biological system are obscure, and that no enzyme-saturation effects are recognizable in the equations. Consequently it was here that the second extension of
thermodynamics, to include the mechanism and kinetics of enzyme-catalyzed reactions, had to start. Rottenberg [ll],Hill [12] and Van der Meer et al. [13] have
generated such an extension by translating the concentration parameters present in
enzyme kinetics into thermodynamic parameters. We shall now first demonstrate the
principles of this for the simpler case of ordinary chemical kinetics.

3. I . The principle

To investigate enzyme kinetics in terms of thermodynamics, it is appropriate to start
with a consideration of a simple chemical reaction [1,4,6,14]:

Here S stands for ‘substrate’ and P for ‘product’. By writing the net velocity of the
reaction as the difference between the forward and the backward reaction:
0=k,

. [ S] - k - , . [PI

(4)

and substituting the chemical potentials of the reactants:
p , = p:

+ R . T . ln[i],

i

=

S or P

(5)