Fundamentals of Biochemistry - Filippovich, Y. B. 1999

Biological Oxidation
History of the Development of Concepts Regarding the Mechanisms of Biological Oxidation Reactions

Understanding such a complex phenomenon as Biological Oxidation is possible only after reviewing the concepts that gradually emerged during the investigation of this problem.

Although more than two hundred years ago (1774–1777) A. Lavoisier considered Respiration to be a very slow combustion of nutrients in the body, similar to the burning of coal, the first attempt to reveal the Molecular Mechanism of biological oxidation was undertaken only in the following century by C.F. Schönbein (1845–1868). He put forward the idea that the activation of oxygen is a necessary condition for biological oxidation processes to occur. Naturally, the specific pathways of this activation seemed fantastic (it was hypothesized that there existed a negatively active form of oxygen, identical to ozone, and a positively active form called antiozone), but the idea itself was fruitful. C.F. Schönbein was the first to suggest that biological oxidation is a catalytic process. He also managed to experimentally prove The formation of H₂O₂ during biological oxidation.

At the turn of the 19th and 20th centuries, our compatriot A.N. Bach and, independently of him in Germany, C. Engler and W. Wild put forward a hypothesis regarding the formation of organic peroxides as The First stage of biological oxidation. In this process, the oxygen molecule was transferred to an activated state due to the Cleavage of its double bond through the "internal vibrational energy" of the oxidizable compound itself, which possessed a multiple bond, and with the participation of Enzymes—oxidases—in accordance with such a scheme, for example:

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The resulting organic peroxides, like hydrogen peroxide, can oxidize other substances under the catalytic action of peroxidase—an enzyme that was already quite well studied at that time:

Hydrogen peroxide can also decompose in another way, with the participation of the enzyme catalase, about which a considerable amount of data had also accumulated during this period:    

Probably for this reason, A.N. Bach attached great importance to the participation of peroxidase and catalase in biological oxidation, believing that the distribution of H₂O₂ between peroxidase and catalase could serve to regulate this process, for example, in plants.

Later on, these enzymes were assigned a more modest role in redox processes.

However, in light of data on active oxygen states, the existence of superoxide ions, and enzymes involved in their METABOLISM—superoxide dismutases—the importance attributed to H₂O₂, catalase, and peroxidase in BIOLOGICAL OXIDATION REACTIONS has begun to grow steadily.

A fundamentally different approach to deciphering the mechanisms of biological oxidation reactions was outlined in the works of V.I. Palladin, and following him, H. Wieland. Based on experiments with respiratory chromogens (by which he meant colorless plant-derived substances capable, in the presence of oxidases, of adding oxygen and converting into pigments that, in turn, could transfer the added oxygen to the oxidizable substrate while simultaneously being discolored), V.I. Palladin was the first (1912) to express the idea that biological oxidation is The transfer of hydrogen from the oxidized substance towards oxygen, resulting in the formation of Water as the end product. V.I. Palladin came to this idea after discovering in one of his experiments that methylene blue, which does not contain oxygen in its composition, can act as a respiratory chromogen by removing hydrogen atoms from the oxidizable substrate:

It became clear that respiratory chromogens are not oxygen carriers, but rather hydrogen acceptors.

V.I. Palladin's concept was confirmed fairly quickly. Thanks to the works of T. Thunberg, J. Sumner, G. Somers, V. McShane, and others, various dehydrogenases were isolated and characterized; these accelerate the oxidation reactions of specific substrates with the participation of Coenzymes that act as acceptors for the hydrogen atoms removed from them.

Thus, at the beginning of our century, two concepts of biological oxidation emerged: the activation of oxygen and the activation of hydrogen. Their confrontation did not last long: in 1925, D. Keilin discovered Cytochromes in the Tissues of A number of insects and subsequently in other aerobic biological objects—the missing enzymes that a few years later allowed the activation of oxygen and hydrogen to be linked together. This was facilitated by O. Warburg's discovery (1928) of cytochrome oxidase, which at the time was named the "Warburg respiratory enzyme." Cytochrome oxidase turned out to be the enzyme that directly activates oxygen, while cytochromes are the enzymes that remove electrons from hydrogen and transfer them to cytochrome oxidase. Thus, THE CONCEPT OF Respiratory Chain enzyme assemblies ensuring biological oxidation reactions arose for the time. In particular, the oxidoreductase chain, the main component of which is cytochromes, was named the cytochrome system:

Enzymes located at the end of such oxidoreductase chains that directly transfer electrons to oxygen were named terminal oxidases.

Later (1947–1966), it was shown that the cytochrome system does not exhaust the list of enzyme systems capable of activating both hydrogen and oxygen, with the subsequent formation of water molecules from them. There are several such systems. The simplest of these, along with peroxidase reactions, is the glycolate oxidase system:

It is present in plants, animals, Fungi, and Bacteria. Glycolate oxidase from spinach leaves is a flavoprotein with M = 270,000 (8 × 37,000). The quaternary Structure of the glycolate oxidase octamer and the Tertiary Structure of the subunits have recently been elucidated; having a diameter of about 10 nm, the octamer, composed of 4 dimers, possesses a cavity 6 nm in diameter. Glycolate oxidase contains flavin mononucleotide (see p. 120) as a coenzyme; through its mediation, glycolic acid is dehydrogenated (hydrogen activation). At the same time, glycolate oxidase is capable of activating oxygen and transferring hydrogen atoms from the reduced flavin mononucleotide to it with the formation of H₂O₂, i.e., it exhibits a flavoprotein oxidase function. The resulting hydrogen peroxide decomposes with the participation of catalase.

Currently, more than two dozen flavoprotein oxidases containing FMN and FAD as coenzymes have been studied (oxalate oxidase, glucose oxidase, L-amino acid oxidases, xanthine oxidase, etc.).

Another type of enzyme system ensuring the direct oxidation of substrates with the transfer of hydrogen atoms to oxygen is represented by copper-containing oxidases. Because concentrated solutions of these enzymes are blue, they are called "blue oxidases." A characteristic representative of this group of oxidases is ascorbate oxidase—a protein with M = 130,000–140,000, containing 8 Cu atoms per molecule and consisting of two equal subunits. It was discovered by A. Szent-Györgyi (1928) and purified by H. Tauber (1938). The equation for the ascorbic acid oxidation reaction is given on p. 171.

The Development of concepts regarding oxidoreductase systems participating in biological oxidation was accompanied by the clarification of their Functions, Classification, and the nomenclature of the enzymes comprising them. Let us recall (see p. 117) that those dehydrogenases which provide direct dehydrogenation of substrates are called primary. In contrast, dehydrogenases that receive H atoms from the reduced coenzymes of primary dehydrogenases (NADH, NADPH, FMN ∙ H₂, FAD ∙ H₂, etc.) or from intermediate acceptors to which hydrogen atoms were transferred from primary dehydrogenases are classified as secondary dehydrogenases. Any dehydrogenases (both Primary and secondary) that transfer hydrogen atoms to specific acceptors are called reductases. As noted earlier, all oxidoreductases that transfer hydrogen atoms or electrons directly to oxygen are called oxidases.

In this regard, attention was drawn to redox systems in which a mediator acts between dehydrogenases and molecular oxygen. H atoms from the reduced dehydrogenase first pass to the oxidized mediator molecule, and only then from it to O₂. Both reactions are accelerated by specific enzymes. The final product of the reaction is water. Quinones and ascorbic acid most frequently play The Role of mediators. In this case, only the reduced coenzyme, such as NADH or NADPH, participates in the interaction with the mediator.

The first evidence of a completely new biological oxidation pathway, unlike any previously studied, was reported by E. André and K. Hou (1932), who discovered a specific enzyme, lipoxidase, in soybean seeds. It accelerated the direct addition of atmospheric oxygen across the double bonds of polyunsaturated Higher Fatty acids. The Use of ¹⁸O₂ and Н₂¹⁸О in experimental studies of similar reactions allowed H. Mason et al. and O. Hayaishi et al., simultaneously and independently in 1955, to prove the existence of a new subclass of oxidoreductases—oxygenases—and to investigate the mechanisms by which they incorporate molecular oxygen into various organic molecules.

According to current data, lipoxygenase has a molecular weight ranging from 60,000 to 190,000 depending on the source Organism, is widely distributed in both the PLANT AND ANIMAL kingdoms, and exists in Multiple molecular forms. In the case of linoleic acid, the reaction proceeds According to the equation:

For the phenol-quinone system, the reaction scheme is as follows:

Lipoxygenases take part in The Biosynthesis of Prostaglandins, Leukotrienes, and thromboxanes from arachidonic acid (see p. 463).    

A new chapter in The Study of biological oxidation was opened by V. A. Engelhardt’s discovery (1931) of the coupling between The oxidation of Organic compounds and the phosphorylation of ADP. While measuring the content of adenosine pyrophosphoric acid—which he tentatively called pyrophosphate—in actively respiring pigeon erythrocytes, he found that its level dropped in a nitrogen atmosphere, but increased back to the initial value in an oxygen atmosphere. Alternating periods of anaerobiosis and return to aerobiosis triggered a new cycle of pyrophosphate "breakdown and resynthesis." This indicated the existence of a mechanism whereby The energy released during oxidation (in the presence of oxygen) is not dissipated, but rather utilized for binding and incorporating inorganic phosphate into the high-energy bonds of adenosine triphosphate: АДФ + Н₃РО_{4 →} АТФ + Н₂О.

Later, V. A. Engelhardt’s ideas were further elaborated by V. A. Belitser and coworkers, who made a major contribution to The Development of the Oxidative Phosphorylation coupling problem during its formative years. In particular, V. A. Belitser introduced the Concept of the oxidative phosphorylation coefficient, defined as The ratio of the number of moles of synthesized ATP to the number of moles of oxygen consumed to drive this synthesis. Based on a series of measurements, this coefficient was found to average around 3.0, indicating that the transfer of two hydrogen atoms (and subsequently two electrons) along the respiratory chain of enzymes is coupled with the synthesis of three ATP molecules; that is, there are three coupling sites between oxidation and ADP phosphorylation.

Research into the subtle mechanisms of oxidation-phosphorylation coupling, which forms the foundation of Bioenergetics, has been ongoing for over half a century and is still far from complete. Below, we will review its current state, which reflects the full complexity of modern problems in biological oxidation.