Table Of ContentPARTICIPANTS
Asterisk denotes contributor to this volume.
*Alberty R. A. * Kasha, M.
r
Arnon, D. I. Klotz, I. M.
*Atkinson, D. E. Koffler, H.
*Barker, H. A. *Kok, B.
Burris, R. Krampitz, L. 0.
*Chance, B. Lardy, H.
*Clayton, R. K. *Lehninger, A. L.
*Colowick, S. P. *Mahler, H. R.
*Cori, C. McElroy, W. D.
Forti, G. Ochoa, S.
*Frenkel, A. *Peck, H. D., Jr.
*Gaffron, H. *Racker, E.
Gest, H. San Pietro, A.
Granick, S. *Siekevitz, P.
Gunsalus, I. C. *Slater, E. C.
»Handler, P. Slonimski, P.
Hunter, E. F. Stadtman, E.
Jagendorf, A. T. Stadtman, T.
*Kalckar, H. *Williams, R.J.P.
*Kamen, M. D. Wolfe, R. S.
Kaplan, N. 0. Wood, H. G.
HORIZONS OF
BBOENERGETI1CS
Edited by
Anthony San Pietro
Department of Plant Sciences
Indiana University
Bloomington, Indiana
Howard Gest
Department of Microbiology
Indiana University
Bloomington, Indiana
Proceedings of a Symposium held at Bloomington, Indiana
October 12-15, 1970
Academic Press l/irJ New York and London 1972
COPYRIGHT © 1972, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY
OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM
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PRINTED IN THE UNITED STATES OF AMERICA
PREFACE
"Bioenergetics," taken in its general meaning, has ramifications in
virtually all aspects of the domain of biological chemistry. Indeed, it has
constituted one of the great currents in the history of the development of
our knowledge of cell metabolism. The discovery by Harden and Young in
1905 of a requirement for inorganic phosphate in the alcoholic fermentation
of yeast represents the tip of a veritable cornucopia, and we can confidently
expect that our present horizons will continue to expand. With the passage
of time, the framework of "energy metabolism" has become clearer through
the efforts and ingenuity of an army of investigators that has included some
of the finest minds in biochemistry. Many elements of the scaffolding of bio-
energetics, however, are still perceived only in dim outline. In recent years,
the vigorous application of new, sophisticated, and powerful investigative
techniques has advanced our understanding of energy metabolism signifi-
cantly but, at the same time, constantly reveals previously unsuspected com-
plexities.
The escalation of research on membrane structure and function from
new points of view and the advent of novel theories of phosphorylation
processes during the 1960's suggested to us that the time was ripe to attempt
a synthesis of knowledge-or, at least, a "stock-taking." Happily, the Ses-
quicentennial Celebration of Indiana University provided the occasion for a
symposium meeting for this purpose. From our planning deliberations, we
finally concluded that progress would best be served if we could gather in-
vestigators with widely different interests and expertise in the manifold
aspects of bioenergetics. Thus, we hoped that the conference would bring
together researchers who did not normally frequent the same meetings and
in this way facilitate "cross-fertilization." We would like to think that this
aim was achieved in some measure, but only time will tell if some new kinds
of progeny will emerge. In the meantime, the reader can form his own judg-
ment from the vast array of topics considered and discussed in the following
pages.
Last, but not least, we note that coupled with the scientific discussions
(held October 12-15, 1970) a program was arranged for the airing of views
IX
PREFACE
on current problems of "Science and Society." Drs. W. D. McElroy and P.
Handler presented addresses in this connection, and Dr. Handler's talk is
reprinted in the Appendix.
We are indebted to Dr. Byrum E. Carter, Chancellor of the Bloomington
Campus of Indiana University, for his encouragement and help in making
arrangements for the symposium, and to The Indiana University Sesquicen-
tennial (Mr. Claude Rich, Director) for generous financial support. Special
thanks are due to Mrs. Virginia Flack and Mrs. Cheryl Fisher for expert
secretarial assistance and manuscript typing.
Anthony San Pietro
Howard Gest
CHAIRMAN1S REMARKS
A. L. Lehninger
Johns Hopkins University
Baltimore, MD.
In the letter of invitation to this Symposium on Bio-
energetics our hosts expressed the hope that our discus-
sions would not become narrowly focussed or polarized.
Rather, they wished us to attempt to synthesize some new
concepts and to crystallize some basic questions amenable
to experimental solution in the 1970!s.
To introduce our first session, which will be devoted
to mechanisms of energy conversion, I should like to make
some general observations on where we are today and to
point out some directions of research from which we may
envision new illumination arising.
There can be no doubt that the most central and over-
riding problem we face today in the study of photosynthe-
sis and respiration is the nature of the chemical and phys-
ical events involved in the conversion of electron trans-
port energy into phosphate bond energy. In both processes
we have chains of electron-carrying proteins which are
fixed in membranes, the structure of which is vital for
their function. In both cases electron transport can lead
not only to ATP formation but also to transport of ions
such as H+ across the membrane, and to changes in the con-
formation of the membrane. We are faced today with a tri-
lemma as represented by the three current hypotheses for
the mechanism of energy conversion.
The chemical coupling hypothesis envisions that ADP is
phosphorylated through a series of consecutive enzyme-cata-
lyzed reactions having common chemical intermediates of a
high-energy nature, similar in principal to the consecutive
steps involved in the substrate-level phosphorylations of
glycolysis, the only difference being that the enzymes con-
cerned are fixed in a membrane rather than in solution in
3
A. L. LEHNINGER
the cytoplasm. One or another of the high-energy chemical
intermediates in this process is postulated to be utilized
in bringing about transport of ions across the membrane
and the characteristic changes occurring in the conforma-
tion of the membrane.
The chemiosmotic hypothesis postulates that electron
transport occurs via a chain of geometrically oriented
electron carriers and generates an electrochemical poten-
tial across the membrane, which is then used to drive the
synthesis of ATP from ADP and phosphate by a physical
coupling to a flow of electrical charges or of protons,
rather than by a chemical coupling process via a common
intermediate.
The conformâtional coupling hypothesis postulates that
electron transport generates a high-energy conformâtional
state of the electron carriers or of the whole membrane,
which is then utilized to drive ATP synthesis. The inter-
mediate high-energy state is postulated to involve the
cooperative interaction of weak non-covalent bonds, such as
hydrogen bonds or hydrophobic interactions. There are some
who view the conformâtional coupling hypothesis as a spe-
cial case of the chemical coupling hypothesis, the only
difference being that conformâtional coupling utilizes as
the energy-carrying intermediate a number of cooperative
non-covalent bonds, rather than a single covalent bond.
As things stand today, both the chemical coupling and
the chemiosmotic models can provide for most of the known
properties of energy-conserving electron transport. But
each has serious weaknesses. The chemical coupling hy-
pothesis suffers from past failures to rationalize the
necessity of membrane structure for oxidative phosphoryla-
tion and to prove the existence of high-energy chemical
intermediates. Perhaps, however, in recent work in the
laboratories of Drs. B. Chance and E. C. Slater we may see
more tangible evidence for high-energy forms of the cyto-
chromes. In its turn the chemiosmotic hypothesis has
suffered from inadequate demonstration of the direct gener-
ation of gradients of protons or electrochemical potential
across the membrane by electron transport. The conforma-
tional coupling hypothesis lacks a convincing molecular
model which can serve as a prototype for the conversion of
conformâtional energy into covalent bond energy. Even the
important reconstitution experiments of Dr. E. Racker and
4
BIOENERGETICS
his colleagues, significant as they are to experimental
analysis of the componentry of oxidative phosphorylation,
are unable to date to resolve this trilemma.
There may however be some other ways of looking at the
problem. I believe much illumination is likely to come
from consideration of the evolutionary and comparative
aspects of the development and differentiation of energy-
conserving redox systems. There are perhaps two major
landmarks in biochemical evolution that are most relevant
to our discussion on bioenergetics. The first, in my opin-
ion, is the evolutionary jump that must have occurred when
the first photosynthetic bacteria arose, possibly following
a long period during which the first prokaryotes employed
strictly anaerobic, fermentative energy-conserving mechan-
isms. This big jump later led, after the build-up of oxy-
gen in the atmosphere, to the origin of aerobic bacteria,
with membrane-linked energy-conserving electron carrying
chains, and ultimately to the origin of eukaryotes. As
we begin to acquire new insight into the background of
these events, we might also derive some insight as to why
the membrane-linked energy-conserving electron transport
systems of photosynthetic and aerobic bacteria and of
eukaryotes, arose from, or in preference to, the simpler,
soluble substrate-level energy-conserving mechanisms char-
acteristic of anaerobic bacteria. Very likely we still
have important comparative lessons to learn from substrate-
level energy-conserving mechanisms.
We should also ask whether ATP has always been the
carrier of chemical energy between energy-conserving and
energy-utilizing processes. Recent work of Drs. H. and B.
Baltscheffsky for example, suggests that pyrophosphate may
once have played such a role and may still do so in some
organisms. But going back even further, it seems quite
possible that there are still earlier energy-rich precur-
sors of pyrophosphate groups. Here we may well give close
consideration to recent experiments which suggest that
respiration-linked active transport in bacteria does not
require ATP or other phosphorylated intermediates and other
experiments showing that Ca2+ transport in mitochondria is
a process that takes primacy over oxidative phosphoryla-
tion. These considerations lead to the idea that perhaps
the original function of membrane-linked energy-conserving
electron transport systems was to preserve internal ionic
5
A. L. LEHNINGER
and nutrient homeostasis by promoting active transport,
and that the capacity to bring about phosphorylation of ADP
was a later evolutionary development.
I think I have now raised more than enough issues and
questions to get us off to a start. I should now like to
introduce Professor H. A. Barker of Berkeley, to begin our
Symposium with a discussion of energy conversion mechanisms
in bacteria.
6
ATP FORMATION BY ANAEROBIC BACTERIA
H. A. Barker
University of California
Berkeley, CA.
The work of Pasteur established the existence of bac-
teria that normally live in the absence of molecular oxy-
gen. Since then a great number and considerable variety
of obligate anaerobes have been described. Like aerobic
organisms, these bacteria obtain energy for growth and
other vital functions mainly by oxidation-reduction reac-
tions. The reductants are mostly common organic compounds,
or sometimes inorganic compounds, which are also used as
energy sources by aerobic organisms. However, the oxidiz-
ing agents of aerobic and anaerobic organisms obviously
differ, since by definition aerobes use molecular oxygen
whereas anaerobes are generally restricted to the use of
organic oxidants such as pyruvate, fumarate or glycine,
although some of these bacteria can use inorganic oxidants,
such as sulfate and carbon dioxide.
The difference in the oxidizing agents used by aerobes
and anaerobes has an important consequence for the nature
of the reactions involved in ATP generation. Most of the
ATP available to aerobic organisms results from the oxida-
tion of reduced pyridine or flavin coenzymes by molecular
oxygen through the mediation of the cytochrome-containing
electron transport chain. Most anaerobes cannot generate
ATP in this way, since they lack both cytochromes and a
high-potential oxidizing agent, like oxygen. Consequently
they are usually dependent on substrate level phosphoryla-
tions to generate ATP. There are undoubtedly some excep-
tions to this generalization. The -possible existence of
several types of anaerobic electron transport phosphoryla-
tion is a subject of considerable current interest and con-
troversy which I shall consider a little later. First I
7
