From the Laboratory of Biochemistry, University of Amsterdam, The Netherlands
The defining episode in my scientific career was my close
association with David Keilin in the
nearly 10 years (1946-1955) during which I was a member of the Molteno
Institute at the University of Cambridge. Not only did my work in
Cambridge determine the direction of my subsequent research, but
Keilin's character, way of working and thinking, and his integrity as
a scientist and human being were a continuing inspiration. I remained
in contact with him until his death, and one of my proudest moments was
when, during his first venture abroad since the War for the conferring of an honorary degree in Utrecht, I was able to show him my laboratory in Amsterdam.
David Keilin was born in Moscow on March 21, 1887, of Polish
parentage; his father was a businessman and small landowner. The family
returned to Warsaw where he graduated from the Górski High School
in 1904. He studied medicine at the University of Liège in
Belgium for a year, but in 1905, being advised that his health would
not stand the strain of medical studies, moved to Paris to study
biology. In 1915, he obtained his doctorate with a thesis on the
biology of insect larvae. In the same year he was invited by G. H. F.
Nuttall to be his assistant at the Quick Laboratory of Parasitology in
Cambridge, England, the forerunner of the Molteno Institute, where he
was to spend the rest of his working life. He was appointed Lecturer in
Parasitology in 1925 and in 1931 succeeded Nuttall as Professor and
Director of the Molteno Institute. He had to relinquish both posts in
1952 upon reaching the compulsory retirement age of 65 but was able to
continue working in the Institute until his death in 1963. He received many honors, including election as Fellow of the Royal Society in 1928 and the award in 1952 of the highest honor of the Society, the Copley
Medal. Many do not understand why he was never awarded the Nobel Prize.
Keilin's paper in the Proceedings of
the Royal Society in 1925 with the
title "On cytochrome, a respiratory pigment, common to animals,
yeast, and higher plants" (1) marked the beginning of studies of what
Warburg later called the respiratory chain (atmungskette), many of us
called the electron transfer chain, and David Green, with some
prescience, the electron transport chain. The story of how Keilin came
upon cytochrome when studying hemoglobin in the horse intestinal
parasite Gastrophilus intestinalis is told in his
posthumously published book (2).
Already 75 years ago there was quite a lot known about biological
oxidations. The word "oxidase" had already been introduced by
Gabriel Bertrand (3) in the 19th century to describe the enzyme
responsible for the hardening of lacquer, now known as laccase. In
1910-1912 Battelli and Stern made thorough studies of the
oxidation of a number of substances by oxygen in the presence of
ground-up tissue and showed the sensitivity of this process to cyanide
(4). They referred to the enzyme responsible as indophenol oxidase from
the color reaction they used to measure its activity. In the early
1920s, Thunberg (5) showed that the oxidation of a large number of
organic compounds such as succinic acid is catalyzed by enzymes, each
specific for its substrate, named dehydrases and later dehydrogenases
by Wieland (6). As is well known, a controversy developed concerning
the mechanism of biological oxidations. Wieland and Thunberg, impressed
by the ability of dehydrogenases to catalyze the oxidation of organic compounds by artificial acceptors such as methylene blue, proposed that
the fundamental action is the activation by the dehydrogenases of
hydrogen atoms, otherwise inert, so that they can react with oxygen.
Warburg, impressed by the presence of iron in respiring cells and the
ability of cyanide both to combine with iron and to inhibit cell
respiration, proposed that the fundamental process is the activation of
oxygen by an iron-containing respiratory enzyme (atmungsferment)
(7).
Keilin's paper made it clear that the electrons derived from the
activation of the hydrogen atoms by the dehydrogenase are transferred
via three hemoproteins, which he named cytochromes a, b, and
c, to an oxygen-activating oxidase. He did not name the
oxidase in his 1925 paper, but in 1927 identified it, on the basis of
its sensitivity to cyanide, with Battelli and Stern's indophenol oxidase and on the basis of its sensitivity to both cyanide
and carbon monoxide with Warburg's atmungsferment (8). Much to
Warburg's chagrin he continued to call it indophenol oxidase and, in
retaliation perhaps, Warburg refused to accept the role of the
cytochromes. This became one of the controversies of the 1930s (see
Ref. 9), matching the vigorous confrontations in this field 30 or 40 years later at the annual meetings of the ASBC.
The basic features of our present picture of the respiratory chain were
established by Keilin and his co-workers in the 1920s and 1930s.
Already in his first paper, he showed that cytochrome b is
the first acceptor of electrons from substrate. Making use of the
exceptional stability of cytochrome c, Keilin and Hartree (10) extracted it from heart muscle. Most importantly, in 1939 (11)
they showed that what had hitherto been thought of as a single
cytochrome a consists of two components that they now called cytochromes a and a3. In contrast to
the other cytochromes, including cytochrome a, cytochrome
a3 combines with carbon monoxide and cyanide and
has, therefore, all the properties ascribed to Warburg's atmungsferment.
By 1939, it was possible to write the respiratory chain as a simple
chain: dehydrogenase That was still the situation when in 1946 I joined Keilin as a
Ph.D. student (rather mature in age by English standards but not in
biochemical knowledge (see Ref. 12)), and more than a half-century
later, this description of the respiratory chain is still valid
although additional electron-transferring components have been added to
it. The first of these was cytochrome c1,
already discovered by Okunuki in 1939 (13) but not generally accepted until Keilin and Hartree in 1955 showed that the absorption band initially ascribed to cytochrome c is derived from two
components, one the classical cytochrome c and the other
Okunuki's cytochrome c1
(14).1
The second addition to Keilin's respiratory chain, proposed in 1948 before cytochrome c1 was accepted, was an
electron-transferring factor acting in the chain between cytochromes
b and c that was irreversibly and specifically
destroyed by aerobic incubation with a dithiol compound, called BAL
(17). After the discovery by Van Potter that the powerful respiratory
chain inhibitor antimycin also inhibits electron transfer between
cytochromes b and c, which he ascribed
(incorrectly as it transpired) to its binding to the factor, he kindly
gave it the name Slater factor (18).
In the late 1950s, F. L. Crane (19) in David Green's laboratory
discovered ubiquinone (coenzyme Q) as a new hydrogen carrier between
the dehydrogenases and the electron transfer chain proper, but it was
not until much later that it was recognized that ubiquinone is also
involved in electron transfer within the respiratory chain (see below).
After many earlier proposals that copper is involved as well as iron in
the oxidation of cytochrome c, this was finally established in the 1960s by Helmut Beinert, using paramagnetic resonance
spectrometry (EPR) (20). Bob van Gelder (21) in my Amsterdam laboratory showed that the cytochrome c oxidase takes up 4 electrons
per molecule, one each into the hemes of cytochromes a and
a3 and two into the copper
atoms.2
The application by Beinert of EPR spectrometry revealed also a whole
new class of electron carriers, the iron-sulfur centers (22). With one
exception, these centers are involved in the transfer of reducing
equivalents from the flavin, by then recognized as a component of all
ubiquinone-reducing dehydrogenases, to ubiquinone, rather than in
Keilin's respiratory chain itself. The one exception was not in fact
discovered by Beinert but by his colleague Rieske and is generally
known as the Rieske iron-sulfur protein (23). The high redox potential,
around about that of cytochrome c1, made it an
attractive site of action of antimycin and a candidate for my old
factor. However, there was no experimental evidence for a reaction with
antimycin, and for many years in Amsterdam we did not know quite what
to do about the Rieske protein until Simon de Vries found that its EPR
spectrum is affected by ubiquinone (24). The breakthrough was made
after Bernie Trumpower (25) showed that, after extraction of the Rieske
protein, antimycin inhibits the reduction of cytochrome
b, instead of its oxidation, as it was supposed
to do if it inhibits the chain between cytochromes b and
c. This reminded me of an old observation by Deul and Thorn (26) in my laboratory that this is exactly what antimycin does after
destruction of the factor, what we called the "double kill" experiment. Sure enough Simon de Vries showed that the treatment I had
used in the 1940s to destroy the factor has a drastic effect on the EPR
spectrum of Rieske's iron-sulfur protein (27). By establishing the
identity of my factor and the Rieske protein, the number of possible
components of the respiratory chain was at least reduced by one.
The double kill experiment is nicely explained by Mitchell's Q cycle
(26) to which I had paid insufficient attention when it was proposed,
despite a friendly letter from Peter saying that it would give him
great pleasure if it turned out that the Q cycle explained the Slater
factor. It does. According to this cycle, there are two possible
entries of electrons from ubiquinol to cytochrome b, one
coupled with the reduction of the Rieske iron-sulfur protein and
therefore susceptible to BAL treatment and one via a separate
antimycin-sensitive ubiquinol-binding site, which (when the cycle is
functioning) operates in the opposite direction by accepting electrons
from cytochrome b. I soon became an enthusiastic supporter
of the ubiquitous Q cycle (29).
I have now got a bit ahead of myself chronologically. Just as is the
case with Keilin's c and a absorption bands, the b band turned out
also to be double, but in this case it is derived from two protoheme
prosthetic groups bound to a single polypeptide chain. The first clue
of the existence of two components came from Britton Chance and was
established in his laboratory in a redox titration by Wilson and Dutton
(30). There was quite a lot of what turned out to be rather cloudy work
on cytochrome b in the 1970s, but the dust settled with Fred
Sanger's determination of its molecular weight via DNA (31), which
told me that it is a two-heme cytochrome (32). Its function was
established by the Q cycle as a transmembrane subunit of
ubiquinol-cytochrome c reductase with the lower potential
heme, denoted b566, accepting electrons from
ubiquinol on the outside of the inner membrane and transferring them to
the higher potential heme (b562) on the inside
of the membrane and eventually to ubiquinone.
Keilin and his students used for their studies of the
respiratory chain a suspension of small particles obtained by grinding heart muscle with sand in weak phosphate buffer that became known as
the Keilin and Hartree heart muscle preparation (33). I do not think
that much attention was given in early studies to the nature or origin
of these particles. Indeed I think that I was the first to show that
they contain about 30% lipid, an accidental observation made when I
was looking for a method of determining the dry weight of the
preparation, since in those days the activity of a respiratory
preparation was expressed by the QO2 (µl of
O2/h/mg, dry weight). When as a newcomer I asked Ted
Hartree how to measure the dry weight of the suspension in the buffer,
he suggested that I precipitate it with trichloroacetic acid,
centrifuge, wash the precipitate, dry it, and weigh it. This I did, but
I decided to speed up the drying process by washing with ethanol. I
found that this decreased the weight by 30%, compared with washing
with water, and changed my definition of QO2 to base
it on fat-free dry weight. I did observe that the ethanol extract was
bright yellow but did not give this any thought, thereby missing the
opportunity of discovering ubiquinone.
The significance of the lipid became clear when at about this time
Albert Claude (34) showed that the site of intracellular respiration is
the mitochondrion and, when the mitochondrion was viewed by thin
section electron microscopy by Palade (35), more precisely in the inner
membrane or cristae. We now recognize that the Keilin and Hartree
preparation consists of submitochondrial particles, or vesicles,
derived from the inner membrane.
No attempt was made by the Keilin school to fractionate the chain apart
from the isolation of cytochrome c. The first success was
obtained by Wainio (36) and Lucile Smith (37) using deoxycholate and cholate, respectively, to disperse the membrane and allow its
components to be separated by conventional ammonium sulfate fractionation. David Green's school (38) importantly expanded this
technique to the separation of what he called four complexes, catalyzing, respectively, the reduction of ubiquinone by NADH (Complex
I) or succinate (Complex II), the reduction of ferricytochrome c by ubiquinol (Complex III), and the oxidation of
ferrocytochrome c by oxygen (Complex IV). I have always
thought it a pity that he gave the name Complex to these multisubunit
proteins, each of which has a clearly defined enzyme function.
In the 1920s Keilin and Warburg envisaged that the function of
the respiratory chain is to catalyze the oxidation of intermediary metabolites by the transfer of electrons derived from hydrogen atoms to
oxygen. That it might have an additional function in ion transport was
suggested in 1939 by Lundergårdh (39), specifically that in plants the
cytochromes act as electron carriers in one direction and as anion
carriers in the opposite direction. The primary function of the
respiratory chain, oxidative phosphorylation, was discovered by
Engelhardt in 1931 (40). Measurements of the stoichiometry (P:O ratio),
made independently in 1939-1940 by Belitzer and Tsibakowa (41) in
Leningrad in the USSR and Severo Ochoa (42) in Oxford in England,
established that phosphorylation must be coupled not, or not only, to
the dehydrogenation of substrate but to electron transfer along the
respiratory chain.
After completing my Ph.D. in 1948 with a thesis on the
succinate oxidase system and a subsequent study of the NADH oxidase system (43), oxidative phosphorylation was the logical next topic for
my research, especially after Al Lehninger's paper on oxidative
phosphorylation coupled to the oxidation of NADH (44). This was a new
field for the Molteno Institute, and given the opportunity by the
Rockefeller Foundation to study in the United States, I spent about 6 months working in Severo Ochoa's laboratory at New York University
learning the new techniques.3
Severo's interests were then mainly on carbon dioxide fixation, but in
the same building, Ef Racker was developing the concept of an acyl
intermediate in the oxidative phosphorylation reaction of glycolysis
(46). Adapting an enzyme assay that he had described, I developed a
procedure that enabled me to measure oxidative phosphorylation between
substrate and cytochrome c (47), the first direct
demonstration of what later became known as "site 2 oxidative
phosphorylation." I continued these studies after returning to the
Molteno Institute and in 1953 published what became known as the
"chemical hypothesis" of oxidative phosphorylation (48) in which,
by analogy with substrate-linked phosphorylation, the energy of
electron transfer is conserved primarily in non-phosphorylated high
energy forms of components of the chain.
Around this time, the two functions of the respiratory chain, ion
movements and oxidative phosphorylation, were beginning to coalesce.
Workers on gastric secretion favored a simple redox pump mechanism,
according to which the secreted protons were those liberated from
hydrogen carriers by transfer of electrons to the cytochromes. In 1951, however, both salt accumulation in plants (49) and gastric secretion
(50) were found to be inhibited by 2,4-dinitrophenol, known to uncouple
oxidative phosphorylation from electron transfer. This focused
attention on ATP instead of electron transfer as the source of charge
separation, and Davies and Krebs (51) proposed in 1951 that "ionic
concentration differences, i.e. osmotic energy ... may
play a role in the synthesis of ATP." Williams (52) proposed that
protons could bring about condensation reactions such as polyphosphate formation.
These concepts were developed by Mitchell (53) into a coherent
hypothesis encompassing a functional link between electron transfer in
the respiratory chain and the translocation in the opposite direction
of protons across the inner mitochondrial membrane, whereby the energy
is conserved as an electrochemical proton gradient. To accommodate
experimental evidence of an H+:e ratio of about 2, in 1966 he introduced the concept of loops in the respiratory chain with two
electrons crossing the membrane from one side to the other followed by
two hydrogen atoms in the opposite direction (54). This very important
concept of the sidedness of the membrane with the specific location of
the electron acceptors and donors was not at first generally accepted,
not only because many did not at first accept (or possibly understand) the precise role of protons envisaged by Mitchell but also because those of us more specifically interested in the respiratory chain knew
that the order of electron transfer originally proposed was wrong.
Mitchell's brilliant proposal of the Q cycle (28), made in answer to
these criticisms, as a description of how the oxidation of ubiquinol by
ferricytochrome c is coupled to the net production of
protons on one side of the membrane and their consumption on the other
side was soon given solid experimental support.
As more became known of the structure of the two large multisubunit
proteins involved in the respiratory chain, namely ubiquinol-cytochrome c reductase and cytochrome c oxidase, as well as
of the ubiquinone-reducing dehydrogenases, such as succinate-ubiquinone
reductase and NADH-ubiquinone reductase, it became clear that their
dimensions are in fact greater than a phospholipid bilayer and that
they are embedded and specifically orientated across the phospholipid
layer, which confirmed in structural terms Mitchell's sidedness concept.
The discovery in the 1960s and early 1970s of more and more
electron-transferring centers in the respiratory chain, particularly the multiplicity of iron-sulfur centers, gave a lot of headaches to
those of us who found even the 1948 sequence of dehydrogenase The real function of the multiplicity of electron transfer centers has
only recently become understood as a result of the structural
information that tells us where the centers are located in the protein,
together with a fundamental increase in our understanding of the nature
of electron transfer. Dutton and co-workers (55) have demonstrated
that, by virtue of electron tunneling, electrons can readily travel
through the protein medium a distance of up to 14 Å between redox
centers but that transfer over greater distances is facilitated by a
chain of electron carriers. Within this distance of 14 Å, rapid
electron tunneling takes place even if the electron transfer is
endergonic, provided that the centers are sufficiently close. It is the
proximity of the redox centers in chains that provides highly
directional electron transfer.
The role of distance between the redox centers in controlling the rate
and therefore the specificity of electron transfer is beautifully
illustrated by the mobility of the Rieske iron-sulfur protein subunit
in ubiquinol-cytochrome c reductase, as shown by the x-ray
crystallographic studies of Berry, Crofts, and their colleagues (56).
In one conformation, stabilized by the ubiquinol inhibitor
stigmatellin, the Fe-S cluster is close enough to the ubiquinol-binding
site to allow its reduction by ubiquinol. In a second conformation, it
is close enough (about 8 Å) to the heme in cytochrome
c1 to permit rapid electron transfer. The
important point is that in neither conformation can both reactions
occur at a suitable rate. For example, in the stigmatellin-stabilized conformation, the iron-sulfur cluster is about 27 Å from the heme. Thus, the reaction mechanism must involve movement of the Rieske iron-sulfur protein. Keilin would have enjoyed this paper. X-ray crystallography of proteins was not strange to him. He supported Kendrew and Perutz in their work and lived to see the solution of the
structures of myoglobin and hemoglobin.
We now know that one of Keilin's cytochromes, cytochrome b,
as well as Okunuki's cytochrome c1, are
subunits of a single protein, ubiquinol-cytochrome c
reductase, and that his cytochromes a and a3 are also bound to a single subunit of
cytochrome c oxidase. Cytochrome c remains a
single polypeptide. In his earlier papers, Keilin often used the
singular "cytochrome" to refer to the cytochrome system, and I
think that he regarded them as acting as a single unit. In 1947, he and
Hartree stated that "the catalysts in the particles, as in the intact
cells, are more or less rigidly held together in a framework that
assures their mutual accessibility and a consequent high catalytic
activity" (33). This idea of an ordered macromolecular assembly,
under the name of the "solid state" model of the respiratory chain,
seems to be winning favor over the "liquid state" model that
envisaged independent free diffusion of the multisubunit proteins in
the membrane and of cytochrome c in the space between the
inner and outer membranes of the mitochondrion (see e.g.
Ref. 57).
In any case, the function of the cytochromes is to transfer electrons.
It is the function of the protons, freed by this removal of the
electrons from the hydrogen atoms of intermediary metabolites, to drive
ion transport and the synthesis of ATP. As Mitchell pointed out in the
conclusion to his Nobel Lecture in 1978, "David Keilin's chemically
simple view of the respiratory chain appears now to have been right all
along." (58).
INTRODUCTION
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
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Fig. 1.
David Keilin (1887-1963).
David Keilin (1887-1963)
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
cytochrome b
cytochrome
c
cytochrome a
cytochrome
a3
oxygen. Strictly speaking the order of
cytochrome c
cytochrome a had not been
established (it could have been reversed), but Keilin was convinced,
correctly as it is now known, that cytochromes a and
a3 are closely associated.
Expansion of Keilin's Respiratory Chain
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
Fractionation of the Respiratory Chain
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
Function of the Respiratory Chain
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
Oxidative Phosphorylation and Topography of the Respiratory
Chain
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
Mechanism of Electron Transfer
TOP
INTRODUCTION
David Keilin (1887-1963)
Expansion of Keilin's...
Fractionation of the...
Function of the Respiratory...
Oxidative Phosphorylation and...
Mechanism of Electron Transfer
REFERENCES
b
factor
c
a
a3
O2 longer than necessary to
accommodate a P:O ratio of 3 in oxidative phosphorylation. At the time
of the International Congress in Switzerland in 1970, I remember that,
in desperation, we proposed double chains.
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FOOTNOTES |
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Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.X200011200
Address correspondence to: ecslater{at}btinternet.com.
1 To my everlasting embarrassment, I had published a paper in 1949 (15) in which I concluded that Okunuki's evidence for the existence of cytochrome c1 was unsatisfactory (see also Ref. 16).
2 That it was much later shown that cytochrome c oxidase contains 3 atoms of copper per molecule is not inconsistent with van Gelder's titrations, because two of the copper atoms are coupled and take up only a single electron.
3 A biographical note on Severo Ochoa is to be found in Arthur Kornberg's "Reflections" (45).
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