THERMODYNAMICS AND RESPIRATORY CONTROL IN AEROBIC

24 Pages·2004·0.21 MB·English

THERMODYNAMICS AND RESPIRATORY CONTROL
IN AEROBIC AND ANOXIC MITOCHONDRIA

FWF Project Application (1989)
Erich Gnaiger, Dept. Zoophysiol., Univ. Innsbruck, Austria

S e l e c t e d C o n t e n t s

Summary 1

Part A: PROJECT PROPOSAL 3

A.1. OBJECTIVES OF THE PROJECT AND PREVIOUS RESEARCH 4
1.1. Bioenergetics and biochemistry: kinetic versus 4
thermodynamic control of flux
1.2. Bioenergetics and nonequilibrium thermodynamics: flux, 5
force and linear phenomenological laws
1.3. Hypoxic and anoxic mitochondria: biochemistry, 7
respirometry and calorimetry
1.4. Previous research funded by FWF 9

A.2. PROPOSED RESEARCH 11
2.1. A new theoretical background 11
2.2. Mitochondrial respirometry under 12
aerobic and microxic conditions
2.3. Calorimetry of aerobic and anoxic 15
mitochondria, cells and small animals
2.4. Electron microscopic mitochondrial structure 17

A.3. LITERATURE CITED 18

SUMMARY

Despite the advances in elucidating the chemosmotic and enzymatic mechanisms of oxidative
phosphorylation, the much debated theory on the dynamics of mitochondrial respiration remains
controversial. Questions on the interface of thermodynamic and kinetic control mechanisms are unresolved.
In the present project experiments will be designed to test the hypothesis that respiratory flux in coupled
isolated mitochondria is under thermodynamic control under a much wider range of conditions than
previously conceived. A new nonequilibrium thermodynamic concept, developed within the frame of the
project, will have important implications on our general view of the dynamics of chemical reactions,
providing a basis for the separation of thermodynamic and kinetic effects.
As empirical tests, studies of isolated mitochondria are proposed using respirometric, biochemical
and direct calorimetric methods under aerobic, hypoxic and anoxic conditions. Electron microscopic analyses
will serve as a control of the quality of isolation of mitochondria from various mammalian tissues and from
euryoxic invertebrates. Correlations between mitochondrial ultrastructure and respiratory flux will be
investigated under incubation conditions exceeding the thermodynamic control range, to test the quantitative
importance of mitochondrial structure-function relations in the control of flux.
P7162-BIO, E. Gnaiger 2

The conceptual and methodological developments in the present project will improve our
understanding of respiratory control in isolated mitochondria and tissues under physiological and
pathological conditions. The proposed concept and the microcalorimetric method have elicited an interest in
collaboration on an international and national basis in a wide range of relevant research, spanning from
regulatory biology, cardiology, issues of pathological long-term hypoxia, to ecologically important aspects of
microbial energetics.
An improved theory on the thermodynamics of irreversible chemical processes is required because
conflicting relations are observed between mitochondrial oxygen flux and phosphorylation potential.
Depending on the incubation conditions, linear or entirely irregular flux-force relations are observed within
identical limits of departure from equilibrium (Jacobus 1985). The outline of a new theory on the dynamics
of chemical reactions is an integral part of the project proposal. The theory will be developed on the basis of
the fundamental equations of diffusion (Einstein 1905). The form of Einstein's diffusion equation, J = -u c F,
where u and F are mobility and force (gradient of the chemical potential) respectively, does not generally
predict linear relations between flow and force. This is due to the fact that concentration, c, is variable.
Einstein's diffusion equation contrasts with the form of the thermodynamic linear phenomenological laws, J
= -L F. The conductivity, L, for diffusion is the product of mobility and concentration. In a linear
concentration gradient the conductivity is a nonlinear function of the force, which is considered prohibitive
for the use of Onsager linear thermodynamics. Therefore, we are forced to drop the nonequilibrium
thermodynamic paradigm of flux-force linearity in the near-equilibrium region.
Next, a form of Einstein's diffusion equation for discontinuous systems is required, which reveals an
important analogy between the concentration difference in Fick's First Law of diffusion and osmotic
pressure. The concentration (activity) difference times RT is the diffusion pressure, linearly related to diffusive
flow by the mobility. Generalized fluxes are linearly related to generalized pressures far beyond the
near-equilibrium range, in contrast to the restricted range of approximate flux-force linearity. For chemical
reactions, the "reaction pressure" is defined in analogy to osmotic pressure, as the product of the chemical
driving force and a concentration term, the generalized "free activity".
A successful application is presented of the concept on "chemical reaction pressure" by providing
the first explanation for the flux-force linearity observed in a kinetically complex gas reaction (data from
Prigogine et al. 1948). More importantly, linear as well as irregular flux-force relations on liver and heart
mitochondria (Jacobus 1985; and original data provided by Dr.W.E. Jacobus) are fully consistent with the new
flux-pressure concept. In all test cases analyzed so far, linear flux-pressure relations were obtained. The
concept offers an explanation of extended flux-force linearities, defines the limits of the linear flux-force
range, and rationalizes irregular flux-force relations in terms of the linear relationship between flux and
pressure of chemical reactions. This provides a significantly improved method, for understanding the
integrated operation of thermodynamic and kinetic mechanisms in the control of metabolic flux.
P7162-BIO, E. Gnaiger 3

THERMODYNAMICS AND RESPIRATORY CONTROL
IN AEROBIC AND ANOXIC MITOCHONDRIA

I. Thermodynamic or kinetic regulation of flux?
The physico-chemical basis of transport and metabolism.
II. Calorimetric and respirometric measurement of flux
in aerobic, hypoxic and anoxic mitochondria.

Part A: PROJECT PROPOSAL

Experimental results on mitochondrial respiration (Fig. 1; Jacobus 1985) indicate that a thermodynamic
explanation in terms of flux-force relations is not generally possible for this fundamental bioenergetic process
(Sections 1.1 and 1.2). As an essential part (I) of the present project I propose a new theoretical background
on nonequilibrium thermodynamics applied to chemical vectorial and scalar processes, to transport and
metabolism (Section 2.1).
The theoretical work began during an FWF research grant at Scripps Oceanographic Institute of the
University of California, La Jolla (Prof. Dr. G.N. Somero) in June 1986. A second FWF Schrödinger
scholarship (1987) made possible the initiation of my collaboration with Prof.Dr.W.E. Jacobus at The Johns
Hopkins University, School of Medicine in Baltimore (Section 4.1). Following our intensive discussions of his
experimental findings, I received for analysis a complete set of original data. In the process of writing the
present grant proposal for continuation of theoretical and experimental investigations, the new
thermodynamic theory evolved further. Now it can in fact support the contention that respiratory control in
liver mitochondria is thermodynamic, albeit not in terms of orthodox non-equilibrium thermodynamic
theory.
The proposal for theoretical work (I) of the scope outlined above may appear too far fetched if not
already supported by theoretical arguments and experimental facts. Therefore, it was necessary to develop the
new concept in full length from first principles (Section 4.1 and appended Theory). Consequently the
significance of the experimental aspect of the project (II) will become immediately clear and requires a brief
description only. It is proposed that coordinated calorimetric and respirometric measurement of flux in
mitochondria in conjunction with biochemical analyses will serve as a test of the range of validity of the
extended thermodynamic theory, and provide new insights into the energetic function of mitochondria under
a wide range of cellular conditions (Sections 2.1 and 2.2). Whole animal calorimetric studies in Innsbruck,
and accompanying 31P-NMR measurements on intact tissues in Dr. Jacobus' laboratory will provide the
important opportunity to test the thermodynamic theory in integrated living systems.
Further complexities of mitochondrial function arise under hypoxia and anoxia. Evolutionary
adaptations to anoxia are well known in euryoxic invertebrates, but direct calorimetric studies on isolated
mitochondria from this ecologically and biochemically distinguished group are not yet available (Section 1.3).
Previous research supported by FWF relates primarily to energetic and regulatory aspects of anoxic
metabolism in whole animal studies (Section 1.4). Therefore, investigations on mitochondria isolated from the
euryoxic oligochaete Lumbriculus variegatus comprise a logical extension of previous projects (Section 2.2).
In collaboration with the Abteilung für Ultrastruktur und Evolutionsbiologie, Universitat Innsbruck
(Prof.Dr.R. Rieger and Prof.Dr.J. Klima), electron microscopic investigations are planned on mitochondrial
ultrastructure. The electron microscopic controls of isolated mitochondria will provide a methodological test
and attempt an analysis of mitochondrial structure-function relations (Section 2.3).
Further collaboration in Austria is planned to apply the developed methods and concepts to clinically
relevant problems of hypoxia (I. Medizinische Universitatsklinik, Universitat Wien; Section 4.2), and to an
ecologically important issue of microbial metabolic control under microxic conditions (Institut für
Mikrobiologie, Universitat Innsbruck; Section 4.3).
P7162-BIO, E. Gnaiger 4

A.1. OBJECTIVES OF THE PROJECT, AND PREVIOUS RESEARCH

1.1. Bioenergetics and biochemistry: kinetic
versus thermodynamic control of flux

Although it is possible to consider the
loss of heat as the 'driving force' of
the process, it must not be assumed that
the rate of the pathway will be governed
by this degree of displacement from
equilibrium.
Newsholme & Start 1973

Flux control analysis today is subject to lively debate. Controversies exist as to the relative contributions of
particular enzyme activities or particular effector concentrations to changes of flux (Crabtree & Newsholme
1987; Kacser & Porteous 1987). Irrespective of enzyme activities, the Gibbs energies of metabolic reactions
under in vivo conditions yield important information on the feasibility or direction of a pathway. The actual
fluxes, however, are thought to be entirely controlled by enzyme kinetic mechanisms and independent of the
magnitude of the free energy changes (Harold 1986). This has become a dogmatic view in biochemistry. "The
assumption that kinetic regulation can and should be discussed in thermodynamic terms - that is in error. ...
The control of metabolic fluxes is in fact kinetic rather than thermodynamic" (Atkinson 1977).
The enzyme kinetic paradigm is deeply rooted in the physico-chemical theory of chemical kinetics
which describes the dependence of chemical flux on activities of reactants, products, and catalysts. The
kinetic rate laws are determined by the specific mechanism of reaction, and the series of elementary reactions
must be known to derive a rate equation. Thus chemical kinetics is a microscopic theory (Alberty & Daniels
1980). In contrast, the macroscopic or phenomenological forces of thermodynamics or the molar Gibbs energies, do
not depend upon mechanism and are not considered important in chemical kinetics. Phenomenological
thermodynamics merely sets some constraints on kinetic rate equations (Denbigh 1971).
In studies of active membrane transport processes in general, and of energy coupling in
mitochondria in particular, the proton electrochemical potential is recognized as a key factor in controlling
vectorial flows and bulk metabolic fluxes. Thus thermodynamic control is inherent in the formulation of the
chemosmotic theory (Mitchell 1961), particularly since the proton electrochemical potential and the
phosphorylation potential are closely correlated (Mitchell 1984). Both, thermodynamic and kinetic
considerations have been advanced in mitochondrial respiratory control analysis, and interpretations continue
to be controversial (Brand & Murphy 1987; Jacobus 1985; Mela-Riker & Bukoski 1985).
Early studies supporting the kinetic mechanism point to the importance of ADP concentration in
respiratory control, responsible for the transition between State 4 (no ADP addition and minimum flux) and
State 3 (high [ADP] and maximum flux) (Chance & Williams 1955; see also Jacobus et al. 1982).
Thermodynamic control was indicated by equilibrium studies of Klingenberg (1961) who related the
phosphorylation potential to the redox state of the electron transport chain. Incorporating an equilibrium
thermodynamic approach, Slater et al. (1973) correlated rates of respiration and the extramitochondrial
ATP/ADP ratio. This correlation was explained as a kinetic control of oxidative phosphorylation by adenine
nucleotide translocation (Slater et al. 1973), or as a thermodynamic control of electron transport via the
difference in low intra- and high extramitochondrial phosphorylation potential (Klingenberg & Heldt 1982;
Soboll et al. 1978). However, the adenine nucleotide translocase may operate at equilibrium since a significant
portion of the ADP within the mitochondrial matrix is bound and the ratio of free ATP/ADP and hence
phosphorylation potential is higher or equal to that in the cytosol (Wilson et al. 1983). In kinetic control not
only [ADP] but inorganic phosphate concentration are recognized as a key substrate regulating catabolic flux
(Brazy & Mandel 1986; Mazat et al. 1986), and the contribution of various steps to the control is a function
of metabolic state (Groen et al. 1982; Tager et al. 1983). Equilibrium thermodynamic analysis, combined with
kinetic considerations point to the phosphorylation potential, the redox state of the mitochondrial NAD
couple, and the amount of cytochrome c as the parameters controlling respiration rate (Erecinsca et al. 1978;
P7162-BIO, E. Gnaiger 5

Nishiki et al. 1978; Wilson et al. 1974). Concentrations of phosphocreatine, inorganic phosphate, ATP and
cellular pH, as measured by 31P-NMR, are related to the phosphorylation potential, work performance and
respiratory flux (Chance et al. 1985; Dawson et al. 1978; Gyulai et al. 1985; Jacobus & Diffley 1986;
Kushmerick 1985).
Since substrate concentrations, [ADP] or [Pi], exert an influence on the in vivo Gibbs energy of
phosphorylation, a thermodynamic contribution to control might be postulated even in the case of results
supporting kinetic mechanisms. However, studies by Jacobus and coworkers provide unequivocal "evidence
against the regulation of respiration by extramitochondrial phosphorylation potentials or by [ATP]/[ADP]
ratios" (Jacobus et al. 1982; Jacobus 1985). When various [ATP]/[ADP] ratios are "clamped" at steady state
by titrating the mitochondria with graded ATPase activities, a near-linear relationship is observed between
phosphorylation potential and respiratory flux, as postulated by nonequilibrium (near-equilibrium)
thermodynamics (Katchalsky & Curran 1965; Prigogine 1967). With increasing net Gibbs energy of the
coupled reaction, the respiratory flux increases (Fig. 1A). However, if the ATPase activity is held constant and
various amounts of ATP are added initially, then the respiratory flux behaves opposite to thermodynamic
expectation. Despite the increase in the net Gibbs energy change of the coupled reaction, the observed
respiratory flux decreases or exhibits a random pattern (Fig. 1B). These results, therefore, do not obey
established concepts of nonequilibrium thermodynamics.

1.2. Bioenergetics and thermodynamics: flux,
force and linear phenomenological laws

This chapter is intended to provide an
introduction to that part of thermody-
namics which is of relevance to bioen-
ergetics. This is the most frequently
misunderstood aspect of bioenergetics,
since most biochemists (like the author)
have little background in physical
chemistry.
Nicholls 1982

Relations between fluxes and forces constitute the central theme of irreversible or nonequilibrium
thermodynamics (NET). The driving force for chemical reactions is the molar Gibbs energy of reaction.
Lack of terminological distinction between chemical energy (an extensive quantity) and chemical force (an
intensive quantity) is confusing; hence I will use the term Gibbs force for molar Gibbs energy, expressed in
units [ J/mol = Jol; 1 kJ/mol = 1 kJol ]. In general, energy is the product of an intensive and an extensive
quantity. Gibbs energy [J] is the product of Gibbs force [J/mol] and amount of substance [mol].
Forces are usually vectors related to vectorial processes or flows. For example, the driving force for
diffusion is the gradient of the chemical potential in units [J/(mol m) = Jol/m = N/mol]. The gradient of
the chemical potential divided by the Avogadro number (6.022045 1023 mol-1) yields the average force per
average particle with the unit of a proper force, newton [N]. Now the fundamental linear force-velocity
equation of statistical mechanics can be applied. At constant friction, the average steady-state velocity, v, of a
particle in the direction of the force is linearly related to the average force per particle, F,

v = -u F (1)

where u is the molecular mobility or the reciprocal value of the frictional coefficient (Glasstone 1948).
In nonequilibrium thermodynamics (NET) it is assumed that, close to equilibrium, "we have linear
relations between the rates and the affinities" (Prigogine 1967), that is between generalized fluxes, J, and
generalized forces, F,

J = -L F (2)
P7162-BIO, E. Gnaiger 6

The generalized fluxes include scalar processes such as the rate of chemical reactions. The corresponding
Gibbs force is a scalar force, an intensive property without direction. By definition, the product of flux-force
conjugates yields the energy per unit time or power [J/s = W],

P = J F (3)

In Prigogine's terminology the product of fluxes and "forces" yields the internal entropy production,

diS/dt = J X

where the "forces", X, are implicitly divided by absolute temperature, X = -F/T. Like negative entropy
production, a positive power can only occur by energy conversion when an output process with positive
power is coupled to an input process with negative power. The net power, integrated over all coupled
processes, must always yield a positive entropy production and a negative power, two equivalent statements
of the Second Law of thermodynamics. A negative power is equivalent to the dissipation of power.
The energetic coupling of input and output fluxes in the metabolic machinery is a central theme of
the power of life. In the process of ATP generation, the output force is the phosphorylation potential, ∆eG
[kJ/mol ATP produced] (subscript e for ergobolic, net generation of ATP; Gnaiger 1987, p.16). If the
process of energy transformation is stoichiometrically coupled, then the normalized input force is the Gibbs
force of the catabolic reaction divided by the stoichiometry of ATP, ∆kG [kJ/mol ATP turnover]p (subscript
k for catabolic, no net change of ATP). The sum of the normalized input and output force is the net Gibbs
force of the catabolic-ergobolic coupled reaction, the catabolic "ATP coupling force", ∆keG (Gnaiger 1987,
p.29).
In accordance with linear near-equilibrium thermodynamics, the ATP coupling force is obtained
experimentally from a plot of the catabolic flux as a function of the phosphorylation potential (Fig. 1A). The
flux is measured as mitochondrial oxygen consumption, JO [nmol O /s], and the extramitochondrial
2 2
phosphorylation potential is calculated from measured concentrations of ATP, ADP and inorganic phosphate
at known pH and magnesium ion activity (Veech et al. 1979). At the intercept with the X-axis the flux is zero,
indicating equilibrium or complete compensation of the catabolic input force by the maximum ergobolic
output force; then the net or ATP coupling force becomes zero (ergodynamic equilibrium; Gnaiger 1987).
The linear regression (Fig. 1A) has the form

JO = a - b ∆eG (4)
2

This can be compared with Eq.(2) written for stoichiometrically coupled mitochondrial oxygen consumption.
Assuming a constant ATP/O ratio, we replace L by L' = L x ATP/O ,
2 2

JO = -L' ∆keG (5)
2

Substituting in Eq.(5) for the ATP coupling force,

∆keG = ∆kG + ∆eG (6)

we get

JO = (-L' ∆kG) - L' ∆eG (7)
2

Comparison of Eqs.(4) and (7) shows that the slope b is the phenomenological coefficient or conductivity, L'
= b, whereas the intercept with the X-axis, a/L', is the negative normalized Gibbs force of the catabolic
reaction, (-L' ∆kG) = a;
P7162-BIO, E. Gnaiger 7

∆kG -a/b = -a/L' (8)

The data on liver mitochondria obtained by the graded addition of hexokinases or creatine kinase (Fig. 1A)
yield an experimental ∆kG value of -62.9 kJ/mol ATP turnover. For reasons not yet known, this is much
lower (less negative) than theoretically expected for the oxidation of succinate to fumarate. The value
expected from thermodynamic calculations is ∆kG= -78 kJ/mol ATP turnover, based on extramitochondrial
reaction conditions.
It is well known that the above analysis is incomplete. 1) Linear flux-force relations are predicted by
NET for the near-equilibrium domain only. For net Gibbs forces of larger absolute magnitude than a fraction
of RT, linearity cannot generally be predicted (Prigogine et al. 1948), and is indeed unexpected (Hill 1977). 2)
Furthermore, the catabolic-ergobolic energy transformation is not necessarily stoichiometrically coupled.
Proton slip or leakage would lead to changes of the catabolic-ergobolic flow ratio as a function of the
output/input force ratio. For the linear domain the Onsager relations can be applied to the coupled processes
of catabolic oxygen consumption and ergobolic ATP production, as discussed by Kedem & Caplan (1965;
see also Rottenberg 1973; Stucki 1982).
However, neither critique 1) or 2) can be invoked to explain the contradiction between NET theory
and experimental results shown in Fig. 1B. The Gibbs forces involved in Fig. 1B are in the same range as in
the linear case in Fig. 1A. Introduction of a coupling coefficient <1.0 for relaxing the stoichiometric
constraint would not lead to the pattern and scatter of data seen in Fig. 1B. Either the fluxes are actually
independent of the forces and exclusively governed by enzyme kinetic mechanisms (Jacobus 1985), or NET
theory is incomplete. A third explanation in terms of established NET theory might be related to changes in
the catabolic reaction and therefore changes in the catabolic Gibbs force. However, the conditions in terms
of catabolic substrates in the experiments (Fig. 1A and 1B) were unchanged and oxygen never became
limiting (Jacobus et al. 1982). The catabolic aspect, therefore, is ruled out as an explanation for the observed
discrepancy, but oxygen and catabolic substrate limitation in general are important under many in vivo and in
vitro conditions. Oxygen limitation of mitochondrial respiration is classically described as the transition to
State 5.

1.3. Hypoxic and anoxic mitochondria: bio-
chemistry, respirometry and calorimetry

When oxygen leaked into the air two
aeons ago, the biosphere was like the
crew of a stricken submarine, needing
all hands to rebuild the systems damaged
or destroyed and at the same time threa-
tened by an increasing concentration of
poisonous gases in the air. Ingenuity
triumphed and the danger was overcome,
not in the human way by restoring the
old order but in the flexible Gaian way
by adapting to change and converting
a murderous intruder into a powerful
friend.
Lovelock 1979

Respiration of isolated mitochondria is not affected by decreasing oxygen pressure except in the 'microxic'
concentration range <1 µmol/l or <0.5% air saturation (Oshino et al. 1974; Sugano et al. 1974; Wittenberg &
Wittenberg 1985). However, due to clustering of mitochondria in sites of high ATP demand and oxygen
gradients within cells, the oxygen dependencies of isolated liver mitochondria and mitochondria in intact
P7162-BIO, E. Gnaiger 8

hepatocytes are different (Jones 1986). On the other hand, Connett et al. (1985a) argue that in red muscle cells
the critical partial pressure of oxygen must be <0.3% air saturation and oxygen is not limiting even at
maximally stimulated oxygen flux.
The respiratory flux in intact hypoxic cells is controlled at constant levels by adjustments of the
phosphorylation potential, resulting in independence of cellular oxygen consumption despite the effect of
lowered oxygen on the mitochondrial redox state (Wilson et al. 1977; 1979a, b). Therefore, "since respiratory
control during hypoxia is only poorly understood, the appropriate conditions for incubations of mitochondria
are not certain" (Jones 1986). An understanding of the control exerted by the phosphorylation potential or by
ADP concentrations is required to interpret the effect on flux of oxygen per se or of variations in the catabolic
Gibbs force as opposed to variations in the ergobolic Gibbs force of ATP production. Independent variation
of the catabolic and ergobolic Gibbs force by induced hypoxia and various ATP/(ADP x Pi) quotients can
help to address the important question under which conditions metabolic flux is controlled by catabolic ATP
generation (drive coupled) or by anabolic ATP demand (load coupled; Gnaiger 1987; Fig. 2). In this context
biochemical, respirometric and calorimetric studies of aerobic, hypoxic and microxic mitochondria will yield
important insights.
Existing discrepancies regarding the importance of hypoxia in vivo can possibly be explained by tissue
differences in cell shape, mitochondrial morphology and distribution, and physiological work loads.
Analogous to the aerobic phosphate loading by graded phosphocreatine splitting at increasing work load
(Chance et al. 1985; Kushmerick 1985; Gnaiger 1987), aerobic glycolysis supposedly plays a regulatory role in
muscular rest-work transitions without occurrence of anoxia at any time or location (Connet et al. 1985b). In
liver, however, glycolysis is known to play an important role in overall energy-yielding metabolism in the
activated state of gluconeogenesis (Soboll et al. 1978), possibly due to local hypoxia (Baumgärtl & Lübbers
1983). Importantly, severe hypoxia occurs under pathological conditions due to impaired oxygen delivery, and
little is known about the role of mitochondrial energy metabolism under these conditions (Schneeweisz 1988;
see Section 5). In tissues sensitive or intolerant to hypoxia, mitochondria suffer from a decay in structure, the
degree of coupling is reduced, and membrane potentials decrease (Zimmer et al. 1985). Rapidly growing
tumor cells have high glycolytic ATP production correlated with a deficiency in mitochondria, as is well
known from cancer tissues (Pedersen 1978). However, even in these neoplastic cells a significant fraction of
total ATP production stems from mitochondrial oxidative phosphorylation, although tumors in vivo may be
less oxygenated than the isolated cells in vitro. It is of current interest to what extent mitochondrial
respiratory control may play a role in the normal to neoplastic transition process and in maintaining or
promoting the transformed state in hypoxic cancer tissue (Pedersen 1978).
In hypoxia tolerant invertebrate tissues, mitochondrial electron transport phosphorylation is not
restricted to aerobic metabolism with oxygen acting as a terminal electron acceptor. Fumarate replaces oxygen
as an electron sink whence phosphorylation coupled electron transport proceeds in anoxic mitochondria. In
contrast to the comparatively poorly understood role of the anoxic function of mitochondria in vertebrates
(Hoberman & Prosky 1965; Ringler & Singer 1959; Sanadi & Fluharty 1963; Wilson & Cascarano 1970),
anoxic mitochondria received much attention in comparative physiology of invertebrates (reviews by Bryant
1970; Cheah 1983; De Zwaan 1983; Gnaiger 1977 1983a; Hochachka & Somero 1984; Saz 1981).
Obviously, the oxygraph method of quantifying respiratory flux in mitochondria fails under anoxic
conditions. Although the direct calorimetric method of measuring metabolic heat flux has been applied to
isolated aerobic mitochondria (Chien & Burkhard 1975; Nakamura & Matsuoka 1978; Poe 1968 1969; Poe et
al. 1967), surprisingly it has not yet been applied to the unique domain of direct calorimetry for measurement
of anoxic electron flux. Biochemical studies of anoxic mitochondria isolated from various invertebrates have
helped to elucidate the mechanism of anoxic electron transport from NADH to fumarate yielding succinate
as an end product (De Zwaan et al. 1981; Holwerda & De Zwaan 1979; Schöttler 1977) and indicated the
mitochondrial localization of ATP production associated with succinate to propionate conversion (Saz &
Pietrzak 1980; Schroff & Zebe 1980; Schulz et al. 1982). The ATP stoichiometries observed in some of these
studies and particularly in studies of aerobic invertebrate mitochondria (Akberali & Earnshaw 1982; Zaba et
al. 1978; Zaba 1983) must be interpreted with care, since improved methods of isolation and more
appropriate assay conditions were developed for fully coupled mitochondria isolated from tissues of various
invertebrates (Ballentyne & Moon 1985; Ballentyne & Story 1984; Burcham et al. 1984; Moyes et al. 1985). In
P7162-BIO, E. Gnaiger 9

the present project biochemical, ultrastructural and cytochemical methods will be applied to test for the
quality of the isolated mitochondrial fraction.

1.4. Previous research funded by FWF

Energy transformation at optimum effici-
ency is the product of the evolution of
energy-coupled systems. The dynamics of
such systems is described by an im-
portant theory of the "thermodynamics
of irreversible processes" (Prigogine,
1967). By definition, conservation of
work in the output reaction is a fully
reversible process. Moreover, cells ope-
rate as isothermal energy converters, so
thermal changes are merely accompanying
features in the transformation of chemi-
cal energy. In this context, it is logi-
cal to replace the terms irreversible
and nonequilibrium thermo-dynamics by
ergodynamics, defined as the theory of
coupled dissipative (irreversible) and
conservative (reversible) energy flow.
Ergo-(work-) dynamics is that branch of
energetics which is concerned with the
transformation and performance of work
in time, the "motion of energy" (from
Greek ergon = work).
Gnaiger 1987

The interest in thermodynamic problems of ergodynamic regulation of metabolic flux developed as a natural
consequence of the need to provide a theoretical foundation to the metabolic and thermochemical
interpretation of biocalorimetric measurements. As a result I have presented a concept on optimum
efficiency of ATP generation under a variety of aerobic and anoxic metabolic states (publications 1-3 listed at
the end of this Section).
The power-economy concept is based on empirically established linear flux-force relations (Section
2.1) without investigation into the thermodynamic basis of linearity. The ergodynamic optimum functions
provide a quantitative assessment of the tradeoff between power and efficiency, particularly in active and
passive anoxia (Box 1).
During the past 10 years the study of aerobic and anoxic metabolism by direct calorimetric,
respirometric, and biochemical methods was the main topic of my experimental research, initiated by an FWF
project to Prof.Dr.W. Wieser, and continued both at the University of Innsbruck (3-8) and in international
collaboration in England and Wales (Institute for Marine Environmental Research, Plymouth; University
College of Wales, Aberystwyth; 9-13) and in USA (University of Maine, Orono; Louisiana State University,
Baton Rouge; University of Colorado, Boulder; 9-12 and 14-17). More recently and primarily due to the
collaboration with Prof.Dr.R. Rieger and his group, some pilot studies focused on the relation between
ultrastructural changes of mitochondria and metabolic functions during transitions between aerobic and
anoxic states (18-20).
The importance of trace amounts of oxygen in the range of 1% air saturation for the energetics of
euryoxic animals was shown in publication (8). Particularly publications (3) and (17) demonstrate the high
potential of biological microcalorimetry combined with respirometry for the study of regulatory mechanisms
in metabolism. These studies remunerated for the efforts in methodological development of the combined
P7162-BIO, E. Gnaiger 10

method of "calorespirometry" (4, 11) which has now become an established technique at the Institute for
Marine Environmental Research in Plymouth, England. International cooperative projects in the USA,
supported by FWF Schrödinger scholarships, provided a basis for two successful NSF equipment grant
proposals for setting up regional centers of biological microcalorimetry and respirometry at the University of
Colorado, Boulder, and at the University of Maine, Orono (Appendices A and B).

Publications

1 GNAIGER E. (1987) Optimum efficiencies of energy transformation in anoxic metabolism. The
strategies of power and economy. In: Evolutionary Physiological Ecology. (Calow P., ed.), Cambridge
Univ. Press, London: 7-36
2 GNAIGER E. (1984) Stoichiometric and calorimetric principles of aerobic and anaerobic energy budgets
and thermodynamic optimization in metabolic economy: rate and power, versus efficiency.
Habilitationsschrift, Univ.Innsbruck: 275 pp.
3 GNAIGER E. (1983) Heat dissipation and energetic efficiency in animal anoxibiosis: Economy contra
power. J. Exp. Zool. 228: 471-490
4 GNAIGER E. (1983) The twin-flow microrespirometer and simultaneous calorimetry. In: Polarographic
Oxygen Sensors. Aquatic and Physiological Applications. (Gnaiger E., Forstner H., eds.), Springer, Berlin,
Heidelberg, New York: 134-166
5 GNAIGER E. (1983) Calculation of energetic and biochemical equivalents of respiratory oxygen
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