IN SEARCH OF NEW BIOLOGICAL DIMENSIONS

ALBERT SZENT-GYERGYI*

When I was a young man, passing through Chicago, Carlson invited
me to give a seminar. After my talk, the secretary handed me a check for
fifty dollars, which was, then, much more than today and seemed to me
a very great lot. I refused to accept it, but the secretary said that I could
not refuse it, as Carlson would feel very badly about it. On my question
as to where the money was coming from, I was told that it was Carlson’s
private money. “Why does he do this?” I asked. “Because he is a prince,”
was the secretary’s answer. It was then I learned that there are princes who
wear their crowns on their heads and princes who wear their crowns in
their hearts. Carlson was one of the latter, and ever since it was of great
encouragement to know that we all can be princes without having been
reared in a golden cradle. Carlson’s cradle must have been very simple

indeed.

Though we do not know what life is, we can distinguish between life
and death, and will not have the least doubt that the cat is alive when it
moves, has reflexes, and excretions. What is underlying these age-old signs
of life is the transformation of chemical energy into mechanical, electrical,
or osmotic work. We do not understand the mechanism of any of these
transformations. This ignorance is in sharp contrast to the brilliant suc-
cesses that biochemistry has achieved in other fields—which suggests that
there is something wrong, somewhere.

Present biochemistry is based on the experience that if we pull a living
system to pieces we arrive at atoms or molecules. So we have to study
atoms and molecules to understand the living organism. No doubt atoms

* The Institute for Muscle Research at the Marine Biological Laboratory, Woods Hole, Massa-
chusetts. This paper was presented as the first Annual Anton J. Carlson Lecture at the University of
Chicago, February 21, 1961.

393
and molecules are the bricks of the building of life, and we have to know
about bricks if we want to understand a building. But will the study of
bricks ever tell us what a Greek temple was, and will the study of molecules
ever tell us what the temple of life is, which is our body? In order to un-
derstand the temple, we must attempt to connect the bricks to higher
units—walls, columns, and the like—in the hope that eventually we may
even approach the sanctuary.

What I propose to do is to throw a fleeting glance on atoms and mole-
cules and then try to connect them. In this first effort we will have to be
very modest, limiting ourselves to first neighbors.

Atoms are built of a nucleus and electrons around it. The paths, orbitals,
on which these electrons can move have different shapes and correspond

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to different “allowed” levels of energy, separated by “forbidden zones.”
We usually symbolize these energy levels by horizontal lines. The lower
ones are occupied, as a rule, by a pair of electrons spinning in opposite
directions, while the higher ones are unoccupied (Fig. 1a). What interests
us biochemists are the outer electrons on the highest occupied levels, which
are involved in chemical reactions. Of the empty levels, the lowest one
will be of the greatest interest. If an electron on the highest occupied orbi-
tal absorbs a photon, then it is “excited” by the absorbed energy, i.e.,
raised to a higher level (Fig. 1b).

Most molecules are formed by atoms sharing a pair of the outer elec-
trons. We can describe the structure of these molecules with fair accuracy,
by the current symbols of chemistry, by various letters (standing for the
various sorts of atoms), while their link we symbolize by dashes. Atoms

Albert Szent-Gyérgyi + New Biological Dimensions
394 Perspectives in Biology and Medicine « Summer 1961
can also be connected by sharing two pairs of electrons, in which case we
write two dashes and call it a “double bond.” If, in a system, every second
bond is a double bond, as in ~-C=C-~C=C-C-, then we call the double
bonds “conjugated.” In this case, the second pair of electrons does not
know whether it belongs to its right or left neighbor. It belongs to both.
In fact, these “‘x” electrons are “delocalized” and belong to the whole
system. Kékulé, who could not then know about 7 electrons, wrote his
benzene formula as in Figure 2a. Today we would rather write it as in Fig-
ure 2b. Anthracene could be written as in Figure 2c. The = electrons, not
belonging to any C atom, but to the whole molecule, we call their orbitals
“molecular orbitals.” These molecular orbitals interest us especially, partly
because many of the most important biological catalysts contain such ex-
tensive systems of =» electrons, partly because the energy levels of these

molecular orbitals can be relatively easily calculated.

 

 

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It was the “solid-state physics” which broke first through this strict in-
dividuality of atoms or molecules. It has shown that if atoms, or mole-
cules, are closely packed with a high regularity (Fig. 32), their electrons
perturb each other’s levels (Fig. 36), which then fuse to one single band
containing many levels (Fig. 3c). Such an energy band (Brillouin zone)
can also be looked upon as a continuous layer of electron gas, extending
over the whole system (Fig. 3d). The system symbolized in Figure 3d will
not conduct electricity since the upper band is empty and the lower band
is full, every atom having contributed two electrons, the maximum num-
ber allowed by quantum mechanics. In such a saturated band, electrons
cannot move, as people cannot move in a room completely filled. The
system could be made conductant either by placing electrons on the
empty band, where these electrons would have a free mobility, or could
be made conductant by taking electrons off from the filled band, creating
“holes” in it.

Another “breakthrough” came about by the study of molecular com-
plexes. In the thirties we find data here and there suggesting that in certain

395
complexes an electron may pass from one molecule to the other. It was
J. Weiss, in 1942, who gave a definite formulation to this “charge-trans-
fer.” In such a transfer an electron passes, as a rule, from the highest occu-
pied orbital of one of the two molecules to the lowest empty orbital of
its partner, the former being thus the “donor,” D, the latter the “acceptor,”
A, as symbolized in Figures 4a and 4b. Under favorable conditions, D
and A can part, D having lost, A having gained an electron. Both of them
will then have an unpaired electron, will thus be what is called a “free
radical,” which can be expected to give a signal in the ESR, the electron
spin resonance experiment. We could write this sequence of reactions as:

D+A=DtA-=Dt++A-. (1)

Oxido-reduction is often defined as an electron transfer. So what, then,
is the difference between “charge-transfer” and “oxido-reduction”? The
difference is an important one. It is this: in most organic molecules the
single orbitals are occupied by pairs of electrons forming a “closed shell.”
In an oxido-reduction, as a rule, a pair of electrons is transferred from one
molecule to another, with the consecutive rearrangement of orbitals,?
leaving behind, thus, two closed-shell molecules. Such a transfer of two
electrons involves a major rearrangement of molecular structure. In
charge-transfer, one electron only is transferred, with the formation of two
radicals, which may remain coupled or may separate. This transfer does
not necessarily involve a major rearrangement of structure.

Does this charge-transfer open any new possibilities for our thinking
about biological processes? It does. Let us imagine, for instance, that an
_ electron would be transferred onto the acceptor from a filled band, as
represented in Figure 3d. Then, a “hole” would be created and the band
would become conductant. Similarly, we could transfer an electron by
charge-transfer onto the empty band, where the electron would have a
free mobility and could thus transport energy. We could also imagine
that the electron, after having been transferred from D to the empty level
of A (Fig. 45), would be transferred from here onto the empty level of a
third and fourth molecule, losing energy in every step. This would be a
sort of “chemistry without chemistry,” and it is this sort of thing which
seems to happen in mitochondrial “oxidative phosphorylation” or photo-

* Michaelis has shown that in many oxydo-reductions the two electrons can pass from one mole-
cule to the other, one by one, going thus through a charge-transfer stage.

Albert Szent-Gyérgyi + New Biological Dimensions

396 Perspectives in Biology and Medicine - Summer 1961
synthesis. Lastly, let us suppose that we transferred an electron to the
empty excited level of molecule A (Fig. 46), then pulled out, by charge-
transfer, one of the two electrons on the highest filled level. Then the elec-
tron could drop down from the excited level into the hole thus created,
emitting its excess energy in the form of a photon. Something like this
may underlie bioluminescence.

I would like to convince you, at this point, that I have not taken you
astray into some abstruse field of quantum mechanics but have been talk-
ing to you on realities. I will make a few charge-transfer complexes. You
will be able to see the transfer of the electron with your own eyes, most
charge-transfer complexes being strongly colored. When wanting to make
a charge-transfer complex of two arbitrary substances, I am faced with a
difficulty. To enable the electron to go from D to A, I must bring the two
molecules into very close proximity, and the attractive forces between

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them may not be strong enough to bring them together. So my first
question is, how can I force two molecules into close proximity? There
are various tricks I can use. I will show you three. Let us take, first, two
water-soluble substances. I will choose two biologically important ones,
riboflavin as acceptor and serotonin as donor, dissolved in water. As you
see, the acid watery solution of their mixture shows the brilliant yellow
color of riboflavin (serotonin being colorless). Now, to push the two to-
gether I need only to freeze the solution. Then the water will crystallize
out, forcing the two solutes into close proximity. As you see, on freezing,
the golden yellow fluid has turned black, like India ink, the free radical of
riboflavin being black. On melting, the electron will return to serotonin,
and the golden yellow color will return. Now let us take two substances
that are soluble in chloroform, like dimethylbenzanthracene and trinitro-
benzene. I will simply pour their faintly pink mixture on filter paper and’
let the chloroform evaporate, leaving the dissolved molecules in close

397
proximity. As you see, a dark purple color appears. I could also dissolve
the two substances in a solvent, like tetrahydrofurane, which mixes with
water. So if I add water now, I dilute the solvent, making the solutes in-
soluble. They have to precipitate together, forming a deeply colored
charge-transfer complex.

I hope that these experiments have convinced you that I did not take
you far away from reality. Now, to convince you that I did not take you
away from biology either, I would like to discuss, briefly, two processes:
mitochondrial oxidation and photosynthesis. As you know, mitochondria
oxidize our foodstuffs and produce ATP by means of their energy released,
“Oxidation” of the foodstuffs means that their electrons become trans-
ferred onto O,. This transfer occurs over a series of intermediary sub-
stances, and so it is customary to talk about the “electron flow” in mito-
chondria. Our question here is: is all this “oxido-reduction,” that is, the
pairwise transfer of electrons, or is it “charge-transfer,” in which the single
electrons go it alone?

There are various pathways in mitochondria. Let us take the most repre-
sentative one, in which the H of the foodstuffs is transferred onto DPN.
The reduced DPN then reduces FMN (flavin mononucleotide), FMN re-
duces a cytochrome, which reduces a second, the second a third, till eventu-
ally the electrons are scavenged by O., which then binds protons, H*’s,
and is turned into water.

Under specific conditions FM can be made to take up one electron,
being reduced to the FMN free radical which gives a signal in the ESR?
(Fig. 5, top) as shown by B. Commoner. If you observe the hyperfine
structure, you find its two sides completely symmetrical, being each other’s
mirror images. As a next step, we mixed FMN and serotonin, making the
same mixture you have seen turn black. The signal was then asymmetri-
cal (Fig. 5, bottom). The reason is simple: the fluid then contained two
different free radicals, of FMN and serotonin, the former substance having
gained and the latter having lost one electron. Then we mixed DPNH
and FMN, expecting again an asymmetrical signal. The signal was com-
pletely symmetrical, identical with that of the FMN radical. There was
no DPN radical. Most probably the DPN radicals “dismutated” to DPN

2 The oxidized form of DPN is DPN?, its reduced form DPNH. For convenience I will write
the oxidized form as DPN and the fully reduced form DPNH2.

3 The curves shown are actually the second derivatives of the signal.

Albert Szent-Gyérgyi » New Biological Dimensions
39 Perspectives in Biology and Medicine +» Summer 1961
and DPNH,, one radical giving an electron to another, both ending, thus,
with paired electrons.

The ESR signal given by FMN (obtained by Dr. Isenberg) can con-
vince you that a charge transfer has taken place; one electron was trans-
ferred without DPN forming a radical. This molecule seems to be built
so that it should be able to give off one electron without forming a free
radical itself. If it would form a free radical, then the positive charge of this
‘radical would attract the electron given to FMN and would not let it go
on its way toward oxygen. From FMN the electron goes through a series
of cytochromes, which are capable of a one-electron change only, so the
whole series seems to be a one-electron business.

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In photosynthesis something similar happens, the difference being that
the electron is raised to the excited level by the energy of the photon ab-
sorbed. The electron then cascades down to its ground level, producing
ATP and DPNH, on its way by its energy released. These two substances
can thus be looked upon as stabilization forms of the very unstable excita-
tion energy. These substances cannot be stored in quantity; thus, a further
stabilization occurs, as is shown by D. Arnon, and the energy of ATP and
DPNH, is converted into the energy of “foodstuffs”—carbohydrates
and fats. We feed on these carbohydrates, then reconvert their energy into
DPNH, and ATP. This takes me to the central theory which is dominat-
ing my research at present. I strongly believe that when ATP and DPNH,
drive the living machine, their energy is reconverted into electronic
energy; that is, they liberate electrons of a high energy, and it, the energy
of these electrons which is driving us, is driving the whole living world.

399
We have been unable to understand the simple signs of life because we
have overlooked this electronic dimension.

If it is this energy of electrons, released in charge transfer, which is
driving the living machine, then it becomes desirable to approach these
changes in a more quantitative, numeric fashion. This can be done by
imagining that we transfer the electron from D to A in two distinct steps.
In the first, we take the electron off from D altogether, into infinity, then
drop it from infinity onto A. The first step consumes, the second liberates
energy. The energy consumed in the first step I have symbolized in Figure
6 by the upward arrow, while the energy liberated in the second step is
symbolized by the downward arrow. The energy gain, or loss, in the total
process is represented by the difference in length of the two arrows. Both
arrows correspond to well-defined physical concepts, the upward arrow
to what is called “ionization potential,” IP, the downward arrow to what

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is called “electron affinity,” EA. Both can be expressed in electron volts
or calories. If we cannot express the energy change simply by IP-EA, this
is because dropping the electron onto A involves various secondary reac-
tions which make EA less definite. So, we can build, only, on the IP, which
is well defined. We can dodge this difficulty by always using the same ac-
ceptor while working with different donors. Then EA will remain constant
and the only variable will be the IP. We biologists can apply this trick be-
cause the final electron acceptor in biology is always the same substance,
O.. It follows, then, that we can express the energy of an electron un-
equivocally by its IP, which can be measured. It can also be calculated,
and such calculations have been done for a host of biological substances
by the Pullmans.‘

According to the theory of Mulliken, if D and A get together, part of
the electron goes over spontaneously while part of it may need the’energy
of a photon for its transfer. The electron will thus absorb a photon. Ab-

*The final result, in this case, was not expressed in terms of IP, but in terms of K or P, which
are linear functions of the IP.

Albert Szent-Gyérgyi + New Biological Dimensions
400 Perspectives in Biology and Medicine + Summer 1961
sorption of photons in the visible range means color, so when you observe
these charge-transfer colors, you actually observe the energy of the elec-
tron transfer. The energy of light, as well as its color, depends on its fre-
quency, so there has to be some direct relation between the IP (or P) of
the donor—that is, the energy needed for the transfer—and the color of
the complex. We can, in fact, directly read the energy change from the
color, Let me demonstrate. I have dried on this paper from right to left, a
solution of indole, chrysene, benzanthracene, 9,10 dimethylbenzanthra-
cene, and phenothiazin. The IP of these substances decreases in the given
order. Now, if I pour over this a solution of trinitrobenzene, which acts
as an acceptor, you see strong colors appearing. Indole gives a yellow color,
indicating that blue light was absorbed, the high energy of this light, about
80 calories, being needed for the transfer. Chrysene is somewhat orange,
benzanthracene is deep orange—light of longer and longer wavelength—
that is, less and less energy being needed for the transfer. The red color of
the complex of the dimethylbenzanthracene indicates a very low ioniza-
tion potential, while the blue color of the phenothiazin complex indicates
that even less energy, about 50 calories, was enough to induce transfer.

Trinitrobenzene, which I used as acceptor, is a rather bulky molecule
which acts with its + orbitals as an acceptor. We might ask, what would
happen if we used a more compact little acceptor, such as the iodine mole-
cule, 1,? As you see, the result is different. Indole colors dark, chrysene and
benzanthracene give no reaction, while dimethylbenzanthracene also gives
a black color. Why, then, did indole give a strong reaction with I, when
it gave a poor reaction with trinitrobenzene? This takes us to a fascinating
field. In a highly conjugated molecule, as is that of indole, the electrons
belong to the whole system but are not equally distributed. The C atom
in position 3 is exceedingly rich in electrons; it has a high electron density.
If we take away this high electron density by converting indole into car-
bazole, the substance does not react with I, any more, though carbazole
has a lower IP than indole. Evidently, indole reacted with I, at this C atom
in a “local” charge-transfer, and it was not solely the IP which decided
the issue, but also the “formal” charge on C3. This is most fascinating be- _
cause it brings into the picture the individuality of the single atoms within
the molecule, and the whole reactivity begins to assume that subtlety
which we find in biological systems.

This reaction has fascinated me also for another reason. The Pullmans

401
have shown that aromatic hydrocarbons, which induce cancer, have two
C atoms in their “K-region,” which C atoms have a very high charge
density. So, if our reasoning is correct, these substances should also give a
black complex with I,, and if this has something to do with cancer, then
other, closely related, substances, which have no carcinogenic activity,
should not give this reaction. This expectation was borne out before when
I showed you that the non-carcinogenic benzanthracene gave no black
complex while its strongly carcinogenic dimethyl derivative did. The ex-
pectation was borne out by a number of other examples. So, for instance,
the non-carcinogenic 1,2 benzpyrene gave no reaction, the carcinogenic
3,4 benzpyrene a very strong one. Further experiments showed that car-
cinogens belonging to quite different groups, like diazocompounds, be-
haved likewise, the carcinogens reacting strongly, the non-carcinogens not
reacting, or reacting poorly. This suggests that production of cancer is, in
some way, connected with a “local” charge-transfer, which may someday
bring us closer to the understanding of this disease.

With Isenberg and Karreman, we also attempted to find out whether -”
some pharmacological reactions might be connected with charge transfer.
We reasoned as follows. Let us take a drug with a unique action. If this
action has something to do with charge-transfer, the substance should also
have unique electronic qualities as donor or acceptor. We chose chlorpro-
mazine, which has such a favorable influence on the symptoms of schizo-
phrenia. We found it to have unique electronic qualities: it is the substance
with the lowest IP known among stable compounds. Serotonin we found
to be an especially good donor. This drug, too, has a unique activity.

So 1 hope to leave you with the impression that a new dimension is
opening up for biochemistry—that of the electrons, a dimension in which
a molecule assumes a new importance as a quantum mechanical frame-
work, Every single atom with its electronic profile acquires a new and
subtle personality. Ideas on which we built, till the present, such as the
mass law action, lose much of their importance, while the whole system,
with its great subtlety, begins, if I may say so, to smell of life. I also hope
to have convinced you that the distance between those abstruse quantum —
mechanical calculations and the patient’s bed is very small, and that further
work on these lines may not only deepen our understanding but may also
help to complete the armory of medicine with which we try to eliminate
human suffering.

Albert Szent-Gyérgyi + New Biological Dimensions

402 Perspectives in Biology and Medicine « Summer 1961