I was reading this article on Albert Szent-Gyorgyi and on page 7 there's this statement:

Now, I thought myself capable of tackling a biochemical problem. I embarked on biological oxidations. At that time a violent controversy raged between O. Warburg and H. Wieland and their followers. The former thought that oxygen activation was the most essential feature of respiration, while Wieland put H-activation in the fore.

This Wikipedia article states that in biochemistry, activation is where biologically active molecules acquire the ability to perform their biological function. What does this mean in the context of the above statement?


2 Answers 2


The answer to this question is to be found in an extensive biography of Warburg published by The Royal Society of London in 1972, written by his former pupil and fellow Nobel laureate, Hans A. Krebs.

The reasons that this is/was difficult to find is that searches for Warburg bring up dozens of papers about the Warburg Effect, and O-, H-activation are obsolete terms reflecting a stage in the study of mitochondrial oxidation (although mitochondria, as such hadn’t been discovered — they were the active components in the particulate fractions used) in which the various haem components of the electron transport chain were in the course of being unravelled. To quote from the publication, starting on p. 648:

Warburg’s next major undertaking was the elucidation of the mechanism of action of the ‘hydrogen activating enzyme’ in biological material. It had been known from the work of Thunberg, Wieland and others, beginning in the first decade of this [the 20th] century, that biological material contains enzymes which catalyse the reduction of methylene blue (and similar synthetic dyes) or nitrophenols to colourless compounds by a variety of substances like succin­ate, malate, citrate, lactate or glutamate. The common feature of these substances is their ability to donate hydrogen atoms and undergo ‘de­-hydrogenation’. Such experiments indicated that O2, the physiological hydrogen acceptor in the process of combustion, can be replaced by other acceptors. During the 1920s it was intensely argued whether the catalytic activation of oxygen, as studied by Warburg, or the catalytic activation of the hydrogen atoms of the substrates, as studied by Thunberg and by Wieland, was the essential feature of biological oxidations. At the earlier stages of the discussion Warburg dismissed the concept of the activation of hydrogen because the experiments of Thunberg and others on which it was based, involved non-biological oxidising agents such as methylene blue or nitro­phenols. He scathingly stated that the oxidation of organic material by derivatives of nitric acid or synthetic dyes is of no biological interest. But he did not properly appreciate that although methylene blue is not a naturally occurring compound, the reactions which Thunberg had studied were enzyme-catalysed processes. Eventually, however, Warburg became im­pressed by the enzymic nature of the methylene blue reduction when E. S. G. Barron demonstrated some methylene blue experiments on red cells to him personally in 1929 while visiting the Johns Hopkins Medical School at Baltimore.

To cut to the punch-line, having abandoned his previous dogmatic position, Warburg went on to identify the riboflavin component (co-enzyme) of the ‘yellow enzyme’ involved, in which the oxidation and reduction is indeed the addition and removal of hydrogen.


Returning finally to the actual question, “what was meant by ‘activation’?”, it was the (re)conversion of the the cofactors (Fe in the cytochromes, the pyridine ring in NAD and NADP, and the flavin ring in FAD) to their higher oxidation state. This differs from the current use of the term activation, e.g. for amino acids in amino acyl tRNAs, or acetate in acetylCoA, where the negative free energy change from breaking a linkage in the activated form drives the formation of a bond with another molecule. However, as redox half-reactions involve calculable free energy changes, it is perhaps ultimately not so different.

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    Apr 5 at 23:00

Warburg-Wieland Controversy

In the years immediately preceding the 1925 publication by Keilin of his seminal papers on the cytochromes, a fierce controversy between Warburg and Wieland was in full flow regarding the nature of biological oxidation (Manchester, 1998).


Wieland and coworkers considered what we would call dehydrogenases were the key to understanding biological oxidation, and that these enzymes 'activated' hydrogen atoms in substrates such as succinate, passing them to a suitable acceptor. In other words they considered removal of hydrogen atoms, catalyzed by dehydrogenases, as central, and that these enzymes act as hydrogen activators.

To this end, they often used a Thunberg apparatus, where the the reduction of the dye methylene blue to a colourless form in the presence of tissue extracts and (for example) succinate could be followed anaerobically. Allowing oxygen to enter the system inhibited the colour change.

As pointed out by Manchester (1998), a key tenet of this mechanism is that oxygen does not directly combine with the organic substrate, and that oxygen atoms are not incorporated into CO2.

The adherents to H-activation were well aware of the role O2 played in respiration, but considered this to be passive, analogous to what happens when O2 is allowed enter a Thunberg apparatus and accepts electrons from colourless (reduced) methylene blue. That is, they saw no requirement for oxygen activation.

Enzymes and 'Specific Activation'

The term 'activation' may require comment, with regards to enzyme catalysis. It was not uncommon in the early days of enzymology to consider an enzyme as 'activator' of a substrate, in the sense of making it more reactive. As late as 1979, for example, Dixon and Webb (1979, p4), defined an enzyme as "a protein with catalytic power due to its power of specific activation" (and argue that cytochrome C should not be considered an enzyme because it acts as a carrier and not by activating any other substance).


Warburg, almost universally accepted as one of the great biochemists, but also renowned for his vituperative attacks on those who did not agree with his ideas (see Gratzer, 1992) was very dismissive of H-activation, which he considered unnecessary and misleading (Hartree, 1963)

Instead, Warburg proposed that all cells have an iron-containing respiratory enzyme, Atmungsferment, (respiratory ferment) which, in conjunction with unspecific surface forces, is responsible for adding oxygen to organic molecules (Keilin, 1966; Hartree, 1963; Manchester, 1998). Thus, Warburg considered oxygen activation as key to understanding respiration.

The Atmungsferment system had one great advantage: it was inhibited by low concentrations of cyanide, a known inhibitor of cellular respiration (Keilin, 1966).

Resolution of the Conflict.

As related by Manchester (1998) and Szent-Gyorgyi (1963), a key experiment that lead the recognition that both H-activation and O-activation are required for respiration was performed by Szent-Gyorgyi. He showed that succinate oxidation by minced tissue extracts could be inhibited by cyanide, but that respiration could be restored by addition of methylene blue. That oxygen activation was necessary could be inferred from inhibition by low concentrations of cyanide (interpreted as knocking out 'O2 activation') but "the dye restored respiration, replacing O2 activation. It was reduced by activated H and then reoxidized spontaneously" (Szent-Gyorgyi, 1963).

But in this regard, the contributions of Keilin, in particular his (re)discovery of the cytochromes, were also critical. Hartree (1963) argues that Keilin's work had two great effects on the elucidation of the mechanism of biological oxidation.

Firstly, the recognition that haem-containing cytochromes were reversibly oxidized and reduced in respiration was a seminal discovery. Among other things, this allowed Warburg to deduced that Atmungsferment contains heme and is inhibited by CO. (We now recognise that a key component of Atmungsferment is cytochrome oxidase).

Secondly, Keilin's experiments lead to the development of the concept of the respiratory redox chain.

To be fair to Warburg, he did concede in his 1931 Nobel lecture that H-activation precedes O-activation: "It must, therefore, be concluded that activation of the combustible substance in the breathing cell precedes the attack of the ferment iron; this corresponds with 'hydrogen activation' as postulated in the theory of Wieland and Thunberg" (Warburg, 1931).

Furthermore, although he originally thought that Atmungsferment catalyzes the direct transfer of oxygen to substrate, he conceded that it is possible "that the iron that reacts with the molecular oxygen does not directly oxidize the activated combustible substances, but that it exerts its effects indirectly through still other iron compounds - the three non-autoxidizable cell haemins of MacMunn, which occur in living cells according to the spectroscopic observations of MacMunn and Keilin, and which are reduced in the cell under exclusion of oxygen" (Warburg, 1931, emphasis is mine).

† Warburg was incensed by the 1927 award of the Nobel prize to Wieland (for work on bile acids) and wrote to his sister that the award had little significance, having been awarded by 'a gang of drunken Swedes' (Gratzer, 1992).

Dixon, Malcolm, Webb, Edwin C. Thorne, C.J.R, Tipton, Keith F. (1979) Enzymes 3rd Edition Longmans, [Available at the internet archive)

Gratzer, W (1992) Warburg's wars Nature 356, 640-641

Hartree, E. F. (1963) David Keilin (1887-1963) Biochem. J. 89, 1-5

Keilin, D. (1925) On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants. Proc. R. Soc. Lond. B. 98, 312–339

Keilin, D. (1966) The History of Cellular Respiration and Cytochrome. Cambridge University Press

Manchester, Keith L. (1998) Albert Szent-Gyorgyi and the unravelling of biological oxidation. Trends Biochem. Sci. 23, 37-40, 1998

Mann, T. (1964) David Keilin (1887-1963) Biogr. Mem. Fellows. R. Soc. 10, 183–205

Szent-Gyorgyi (1963) Lost in the Twentieth Century Annu. Rev. Biochem 32, 1-15

Warburg, O. (1931) The Oxygen-Transferring Ferment of Respiration. Nobel Lecture


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