Oxidizing Enzymes

OXIDIZING ENZYMES, a group of enzymes that drive oxidation processes occurring in plant and animal cells both during their life and for some time after the destruction of protoplasm. The most important biological oxidation process of the living cell is respiration, in which organic substances are oxidized by atmospheric O₂ to final breakdown products (CO₂ and water). Lavoisier already pointed out that this process is analogous to combustion. But whereas combustion can occur only at very high temperatures of organic substances, respiration takes place at relatively low temperatures. Therefore, it must be assigned to the group of phenomena very widespread in nature, usually called "spontaneous oxidation" or "slow combustion". These processes proceed at a noticeable rate only in the presence of special accelerators-catalysts. The necessity of such catalysts-oxidizing enzymes-in the living cell was pointed out by Schonbein. He believed that without these enzymes "animals would suffocate in an ocean of pure O₂ as in an empty space." For the first time, an oxidizing enzyme was isolated by the Japanese scientist H. Yoshida from the sap of the lacquer tree, which is why it was named laccase. G. Bertrand, who studied laccase in detail, showed that similar enzymes exist in other plant objects. He united all these enzymes that oxidize in the presence of atmospheric O₂ under the general name of "oxidases". Bach, together with R. Chodat, subjected these enzymes to detailed investigation. Based on the data obtained, he constructed a theory according to which the action of oxidases is based on the activation of atmospheric O₂. This produces intermediate peroxide compounds of organic substances and hydrogen peroxide. The latter, in the presence of a special oxidizing enzyme, "peroxidase", can be used by the living cell for further oxidation of organic substances. However, neither oxidase nor peroxidase can oxidize carbohydrates, proteins, or fats. Bach and Chodat, through a series of careful experiments, proved that the action of these enzymes extends only to the following substances: phenols, aromatic amines, and hydroiodic acid. None of these compounds is the material oxidized in the process of respiration. Consequently, the presence of oxidases in the cell cannot yet determine the process of respiration. The opinion was even expressed (P. Portier) that oxidases are not "respiratory enzymes" at all and that their role is reduced only to a protective action, to the formation of substances preventing the penetration of microorganisms into wounded tissue. But this point of view turned out to be incorrect, and at the present time, the prominent role played by oxidases in vital oxidation has been established with complete clarity. Respiration is a complex process consisting of a number of separate interconnected and coordinated chemical reactions. Oxidase-mediated oxidation is only one of the links in this long chain of phenomena. Therefore, it is not possible to reproduce the entire phenomenon with oxidases alone. Another extremely important link of the respiratory process is oxidation-reduction reactions occurring within the living cell. M. Traube already showed that oxidation can be carried out not only at the expense of free atmospheric O₂, but also at the expense of bound O₂, in particular water O₂. The simplest example of such hydrolytic oxidation can serve the oxidation of alkali metals, proceeding at the expense of water hydroxyls with the release of the corresponding amount of H in the form of gas: 2Na+2H₂O = 2NaOH + H₂. But this kind of reaction can occur only when the oxidizing body has a sufficient reserve of energy to decompose water by itself. Usually, oxidations at the expense of water elements occur through coupled reactions, in which out of two bodies incapable of decomposing water separately, one binds water O₂ and the other H. For example, metallic zinc in the absence of water is not oxidized at all by atmospheric O₂ and does not decompose water by itself. But if zinc is shaken with water and O₂, the metal combines with the hydroxyl of water, giving zinc hydroxide, and the H of water with an O₂ molecule forms hydrogen peroxide. Another example of the same kind of oxidation-reduction reactions proceeding at the expense of water elements can serve the Cannizzaro reaction (see Cannizzaro reaction), which reduces to the simultaneous reduction and oxidation of aldehydes proceeding in the presence of alkali. In this case, one aldehyde molecule is oxidized by the water hydroxyl into the corresponding acid, while the other is reduced by hydrogen into an alcohol. In vitro, this reaction can proceed only in an alkaline solution, but in the living cell, it is carried out even in a neutral medium due to the presence of special oxidation-reduction enzymes (oxidoreductases). For the first time, the action of these enzymes was discovered with complete definiteness by F. Schardinger in milk. Schardinger showed that a mixture of formaldehyde and methylene blue in an aqueous solution is discolored in a few minutes by fresh milk. Boiled milk remains inactive. Subsequently, it was established that we have here an oxidation-reduction process proceeding at the expense of water elements. In this case, the aldehyde is oxidized to the corresponding acid, and methylene blue is reduced to a colorless leuco compound. Methylene blue and formaldehyde can be replaced by other substances (e.g., other aldehydes, amino acids, purine bases, on the one hand, and various dyes, nitrates, thio compounds, on the other). The character of the reaction does not change from this. Bach, on the basis of a number of considerations, called this milk enzyme "perhydridase". Similar enzymes were found in other plant and animal materials. At the present time, it can be assumed that oxidoreductases are present in all living cells without exception. The biological role of these enzymes is enormous. It is precisely thanks to them that the primary oxidation of various organic substances (in particular respiratory materials) is carried out in the living cell. Carbohydrates, fats, proteins, and their breakdown products cannot be directly oxidized by atmospheric O₂. This oxidation proceeds according to the scheme indicated above at the expense of water hydroxyl. However, as we have seen earlier, this kind of oxidation is carried out only when a reduction process occurs simultaneously with it, by means of which the H of water is removed. In the living cell, very diverse compounds can play this role of traps, hydrogen acceptors. In particular, during fermentation, various carbohydrate breakdown products (for example, aldehydes) undergo reduction. As a result of this, reduction products-alcohols, methane, and even in some cases H-are necessarily formed in the indicated process. Atmospheric O₂ plays no role here, and therefore a number of fermentations can be carried out even in its complete absence. The situation is different in the process of respiration. Here, hydrogen acceptance is carried out by a special mechanism in which oxidases play a far from minor role. As numerous studies by V. Palladin and other authors have shown, substances that can oxidize with the assistance of these enzymes—polyphenols—are present in plant and animal cells along with oxidases. Thus, aerobic oxidation and, in particular, cell respiration take place according to the following scheme: atmospheric oxygen under the influence of oxidases oxidizes "respiratory chromogens"—substances of the polyhydric phenol type. In this case, quinone-like compounds are formed, the so-called "respiratory pigments". The latter possess the ability to bind water H and back-reduce into chromogen, while water hydroxyl goes to the oxidation of various organic substances (carbohydrates, amino acids, etc.). The oxidation of these substances and the reduction of pigments coupled with it is carried out in the living cell only thanks to the presence of oxidoreductases. Apparently, playing a significantly greater role than the polyphenols indicated by V. Palladin as an acceptor and carrier of oxygen both in plant cells (yeast, bacteria) and especially in animal cells is the hemin derivative—histohaematine discovered by Mac Munn, or cytochrome (Keilin), contained in all cells without exception that possess aerobic metabolism. Cytochrome easily passes from the oxidized form to the reduced one and back. Since these forms differ in their spectral properties, their mutual transition—and thus, to a certain extent, cell respiration—can be monitored directly by the eye, without even disturbing the integrity of the cell. Cytochrome is easily reduced by the hydrogen of organic molecules activated by dehydrogenases (see below), or, if one adheres to the scheme of hydrolytic oxidation, by the hydrogen of water under the influence of oxidoreductases.

In turn, the reduced cytochrome, under the influence of a special oxidase differing in some properties from ordinary phenolases, is again oxidized to its initial form by atmospheric oxygen. A system composed artificially of its components, cytochrome + oxidase, revealed in Keilin's experiments a complete similarity to the natural respiratory enzyme. Consequently, two categories of enzymes take part in the respiration process: firstly, oxidases—enzymes that cause the formation of H acceptors (respiratory pigments), and secondly, oxidoreductases—enzymes that accelerate oxidation-reduction processes proceeding at the expense of water elements. This division of oxidizing enzymes into two groups appears entirely justified from both a chemical and biological point of view. However, not all authors agree with the respiration scheme outlined above. In particular, Wieland (H. Wieland) completely denies the activation of atmospheric O₂. In his opinion, any oxidation of organic substances in a living cell begins with the hydration of these substances, with the addition of a whole water molecule. Subsequently, hydrogen is removed from the hydrates thus obtained, dehydrogenation takes place, as a result of which we obtain compounds more oxidized than the initial substances. As an example of such process, Wieland cites the reaction of oxidizing CO to CO₂ in the presence of palladium black as a hydrogen acceptor. CO+H₂O=H·COOH→CO₂+H₂ (acc.). The main point of the entire process as a whole is, according to Wieland, dehydrogenation, proceeding at a noticeable rate only in the presence of specific enzyme catalysts whose action is directed toward the liberation of active H. If hydrogen thus separated from the hydrate molecule, it can then easily be captured by any acceptor, whether methylene blue, aldehyde, or even gaseous O₂. Consequently, no activation of air O₂ by oxidizing enzymes is required or occurs. The action of oxidases, as well as oxidoreductases, is directed toward the H of hydrates, toward their dehydrogenation. Therefore, Wieland denies the existence of oxidases as an independent group and proposes to unite all oxidizing enzymes under a single name "dehydrases." To confirm his opinion, Wieland performed a series of experiments aimed at showing that typical oxidases promote the oxidation of the substrate even in the absence of gaseous O₂ with methylene blue as the H acceptor. Conversely, oxidoreductases produce oxidation associated with the absorption of atmospheric O₂. However, in his experiments Wieland did not deal with physiologically pure enzymes, and the preparations he used could simultaneously contain representatives of both the first and second groups of oxidizing enzymes. Further studies showed that when carefully purified enzymatic preparations are used, it is not possible to repeat the indicated experiments, which of course significantly reduces the persuasiveness of Wieland's arguments. The noted disagreement in the fundamental views on the nature and biological significance of oxidizing enzymes creates very strong difficulty in the study of this branch of enzymology. A diametrically opposite point of view in relation to Wieland's views is occupied by Warburg, who sees the primary moment of the action of respiratory enzymes in the activation of oxygen. This activation takes place under the influence of catalytically active organic compounds (and partly perhaps also free ones) of heavy metals, mainly iron, to a lesser extent also copper, manganese, etc. Of biologically dominating significance as respiratory enzymes (in the sense that Warburg uses this term, i.e., oxygen activators) are complexes of iron with pyrrole compounds of the hemin type, in turn bound to certain organic bases (similar to how, for example, in hemoglobin hematin is bound to the basic protein globin). With exceptional elegance of experiments, Warburg even managed to obtain the absorption spectrum of the respiratory enzyme without isolating it as a chemical individual from the cell (which does not yet seem possible). This spectrum, giving the main absorption band in the region of about 4,300 Å, almost exactly coincides with the spectra of artificially obtained hemin a compounds (only slightly shifted toward the red side). Undoubtedly, the explanation of the true mechanism of oxidative processes in the cell should be sought in the synthesis of the extreme views of Wieland (exclusive activation of hydrogen) and Warburg (exclusive activation of oxygen). The overwhelming majority of modern researchers lean toward this, and Warburg himself in one of his very latest works also recognizes "substrate activation" as one of the possible factors of biological oxidations. Such a synthesizing view essentially boils down to the fact that the main pathway of oxidative processes in the cell is as follows: the activated hydrogen of organic molecules (or their hydrates, according to Wieland, or simply the hydrogen of water in the scheme of hydrolytic oxidation) through the intermediate participation of acceptors or hydrogen carriers (of the respiratory pigment or cytochrome type) reacts with oxygen activated by oxidases or Warburg's respiratory enzyme. However, this pathway is not the only one. In a number of cases of biological oxidations, the formation of hydrogen peroxide has been established. This can happen only if the hydrogen acceptor is molecular, unactivated oxygen: 2H+O₂=H₂O₂, whereas active, atomic oxygen would give the formation of water. Apparently, one has to reckon with the formation of hydrogen peroxide in most biological oxidations. At the same time, the oxidizing enzymes themselves are very sensitive to hydrogen peroxide, being rapidly destroyed by it. Hence the significance of the ubiquitous enzyme catalase (see), which decomposes hydrogen peroxide into water and molecular oxygen, which can again be used by the cell, becomes clear. In addition, the peroxide can also be decomposed by peroxidase with the cleavage of active oxygen, also going into new oxidations. Recent work (1931) established a fact of extremely great importance: it turned out that the last two enzymes, catalase and peroxidase, which are components of the general oxidative system of the cell, are, like Warburg's respiratory enzyme, iron-containing pyrrole derivatives close to hemin and giving a characteristic absorption spectrum with a main band at 4,200 Å. At the same time, the intensity of this band runs approximately parallel to the activity of the enzyme preparation. The hemin content even in the purest preparations is extremely small, on the order of 0.1–0.6%. Obviously, if the enzymatic action is inherent precisely to the indicated hemin derivatives (and this can already be asserted with almost complete certainty), their enzymatic activity (distinguished moreover by high specificity) turns out to be approximately 10⁷ times higher than that of ordinary iron-containing pigments (hemoglobin and its derivatives). The nomenclature and classification of oxidizing enzymes is extremely confused and contradictory. This is due mainly to two circumstances: firstly, the nomenclature of this group of enzymes is not subject to any definite principle and is rather random in nature. Therefore, often the same enzyme appears in the literature under several different names. Secondly, only a very limited number of works on oxidizing enzymes were carried out with physiologically pure preparations. In the majority of cases, researchers dealt with complex autolytic mixtures, tissue mashes, acetone-killed cells of microorganisms, or even simply freshly cut animal organs. It is clear that in such objects the oxidation of a particular substance may depend on the joint action of a whole complex of enzymes. However, it was usually attributed to one special enzyme specific for a given substrate. For a number of cases, it has already been possible at present to show that this is incorrect. The indicated "enzymes" are not individual, but represent a mixture of oxidases and oxidoreductases, the joint action of which causes one or another oxidative reaction. Therefore, in the subsequent presentation, the classification of oxidizing enzymes is based on their division into two groups: 1) oxidases and 2) oxidoreductases. But since individual enzymes have not yet been isolated from a number of mixtures and we know so far only their total action, in some cases it is temporarily necessary to preserve the old terminology and single out these enzymes into a special mixed third group. Group I, oxidases (true oxidases, direct oxidases, aerooxidases, chromooxidases)—enzymes causing the formation of respiratory pigments. The basis of the action of these enzymes is the activation of oxygen. Belonging to this group are: 1. Phenolases—enzymes that oxidize various polyhydric phenols, aromatic amines, and hydroiodic acid with atmospheric oxygen. These enzymes are very widely distributed in both animal and plant cells, where they can be detected using the reaction with pyrogallol.

However, the isolation of phenolases in the form of purified preparations is associated with significant difficulties due to their instability. Therefore, obtaining active preparations is only successful when using certain, mainly plant, objects (mushrooms Russula delica). Based on a number of data, Bach and Chodat suggested that phenolase consists of two parts, of which one activates the O₂ molecule with the formation of peroxide, and the other accelerates the oxidative action of the hydrogen peroxide arising thereby. The first component was named oxygenase, and the second, which activates hydrogen peroxide, peroxidase. Schematically, the action of phenolase can be depicted by the following equations: 2) R-/\ - OH H-O R-/\ - O +

| +2H_aO KJ-on n-o \J-o Peroxidase was discovered in a number of plant and animal objects even in the absence of oxygenase as an independent enzyme. 2. Peroxidases are the best-studied oxidizing enzymes. Their action is completely analogous to the action of phenolases. But whereas the latter activate atmospheric O₂, peroxidases effect the oxidation of phenols, amines, etc. solely at the expense of hydrogen peroxide. Peroxidases are very stable with respect to various external influences and therefore they can be easily obtained from various plant and animal objects. The roots and sprouts of higher plants contain an especially large amount of peroxidase. From horseradish root, Willstätter (R. Willstätter) and his pupils (R. Kuhn) obtained the purest preparations of peroxidase, which made it possible to approach the elucidation of the chemical nature of this enzyme. It is very interesting that the coloring matter of blood, oxyhemoglobin, possesses peroxidase action. But even when it is isolated in a chemically pure form, its action is incomparably weaker than the action of plant peroxidases. 3. Tyrosinase is an enzyme that oxidizes, with the aid of free oxygen, one of the protein breakdown products—tyrosine (p-oxyphenyl-α-aminopropionic acid). Under the action of tyrosinase, a colorless aqueous solution of tyrosine in the presence of atmospheric oxygen becomes colored red, then brown, violet, and black. After this, an ink-black precipitate of melanin falls out, and the liquid becomes colorless again. The reaction proceeds with the elimination of ammonia and CO₂ (deamination of the alanine group). Tyrosinase is less widespread than the first two indicated enzymes. However, it can be detected in a number of plant and animal objects. Potato peel is especially rich in tyrosinase. The oxidation of tyrosine and the formation of melanin from it represent a combination of successive reactions. Therefore, tyrosinase is hardly an individual enzyme. Most likely, it represents a mixture of enzymes, but since oxidases play the dominant role here, it is possible to assign it to the first group of oxidizing enzymes. In addition to what has been said, one must note yet another group of oxidases named by Dubois (R. Dubois) luciferase. The luminescence of a number of lower marine animals, insects, and bacteria is caused by the action of these enzymes. According to the data of the indicated author, an enzymatic oxidation of a special substance, luciferin, by atmospheric oxygen takes place here. However, this reaction is far from being fully studied (see Luminescence). Group II - oxidoreductases (reductases, dehydrases)—enzymes that accelerate oxidation-reduction processes proceeding simultaneously at the expense of water elements. 1. Perhydridase (Schardinger's enzyme) is an enzyme of milk, from which it can be obtained in the form of a purified preparation by precipitation with acetone. Perhydridase simultaneously effects the oxidation and reduction of various organic and inorganic substances. In particular, various aldehydes, amides, and amino acids are oxidized in its presence. The reduction of such substances as, e.g., methylene blue, nitrates, thio compounds, etc., must necessarily be coupled with this. Oxidation and reduction proceed at the expense of water elements, and therefore the reaction can also take place in the absence of gaseous O₂. Atmospheric O₂, on the contrary, even slows down this reaction, since it gradually destroys the enzyme. By virtue of the fact that perhydridase can simultaneously oxidize and reduce aldehydes (Cannizzaro reaction), some authors regard this enzyme as a special case of aldehydeases. 2. Aldehydeases (aldehydrases)—enzymes catalyzing the Cannizzaro reaction. The action of these enzymes was first discovered during the oxidation of salicylic aldehyde into salicylic acid. Therefore, the indicated enzyme initially received the name salicylase. However, it was subsequently established that salicylase can also act on other aldehydes. In this process, along with the oxidation of one aldehyde molecule into the corresponding acid, another molecule is reduced to an alcohol. The reaction successfully proceeds even in the absence of atmospheric O₂, since it proceeds at the expense of water elements. Along with the reduction of the second aldehyde molecule, other substances can also be reduced, in particular, as Neuberg (C. Neuberg) showed, ketones, nitro compounds, thio compounds, etc. Aldehydeases are extremely widespread in both the animal and plant worlds. Animal liver and yeast are especially rich in them. The indicated enzymes play an exceptionally important role in substance metabolism in the living cell. Unfortunately, they are still very poorly studied. 3. Aldehyde mutases—enzymes catalyzing the reaction termed the "internal Cannizzaro" (Neuberg). In this reaction, the simultaneous oxidation and reduction of two atomic groups of one and the same molecule is carried out, proceeding at the expense of water elements. The best-studied example of this reaction can serve as the conversion of methylglyoxal into lactic acid: CH₃ CH₃ | | CO + H₂ -> CHOH COOH According to Neuberg, this reaction is carried out thanks to the action of an enzyme which he called ketone-aldehyde mutase (Dakin's glyoxalase). Group III of oxidizing enzymes (mixed group). These include: 1. Alcohol oxidases (alcohol dehydrases)—enzymes that oxidize alcohols into the corresponding aldehydes. These latter are then converted into acids by aldehydeases. Oxidation is carried out at the expense of atmospheric O₂, but since oxidoreductases are part of these enzymes, oxidation can also proceed in the presence of such H acceptors as, e.g., methylene blue. Alcohol oxidases were first obtained by Buchner (E. Buchner) in the form of acetic acid bacteria killed with acetone. Later, this enzyme was found by Battelli (F. Battelli) and Stern in animal tissues. Horse liver is especially rich in it. 2. Acid oxidases (acid dehydrases)—enzymes that oxidize hydroxy acids. They were first discovered by Battelli and Stern in muscles and other tissues of animals. The enzyme acting on succinic acid (Battelli and Stern's succinoxidase or Thunberg's succinodehydrase) underwent the most detailed study. 3. Purine oxidases (purine dehydrases)—enzymes that oxidize breakdown products of nucleic acids, purine bases. Adenine and guanine arising during the hydrolysis of nucleic acid are converted into hypoxanthine and xanthine under the influence of hydrolytic enzymes. Purine oxidases act on these bodies. E.g., xanthine oxidase oxidizes hypoxanthine into xanthine, and the latter into uric acid. Uric acid under the influence of uricase is further oxidized into allantoin. Purine oxidases have been found only in animal organisms.

A. Oparin, E. Engelhardt. The study of cells for the presence of oxidizing enzymes in their protoplasm is of great importance both in the clinical laboratory and in the pathological-histological study of tissues. It is used mainly for the detection of cells of the bone marrow (myeloid) type, which, unlike elements of a lymphoid and histioid character, contain oxidizing enzymes in their protoplasm. The need to differentiate cells using reactions for oxidizing enzymes in clinical practice arises when it is necessary to determine the type of leukemia in cases where the latter is based on an increase in the number of white elements that are morphologically unclear regarding their nature. Reactions for oxidizing enzymes are performed in smears, e.g., of blood, best fixed in formalin vapors, and in sections made on a freezing microtome; tissue pieces are also best fixed in formalin. Of the reactions for oxidase, the most widely used is the Winkler-Schultze reaction (Winkler, Schultze), the basis of which is the exposure of tissue to solutions of α-naphthol and dimethylparaphenylenediamine. Following Graff's suggestion, this reaction is commonly called the Nadi reaction (Nadi-Reaktion). According to Winkler-Schultze, the solutions of naphthol and dimethylparaphenylenediamine are used separately; according to Graff, in a single common mixture. According to the Winkler-Schultze method, the α-naphthol solution is prepared such that 1 g of α-naphthol is dissolved in 100 cm³ of distilled water, bringing the latter to a boil and adding pure caustic potash drop by drop until the α-naphthol dissolves (usually about 1 cm³ of caustic potash is sufficient). Dimethylparaphenylenediamine is dissolved in an amount of 1% in distilled water at room temperature. Smears or sections are immersed for several minutes first in the naphthol solution and then directly into the dimethylparaphenylenediamine solution; then rinsed in distilled water. Smears are dried and examined with an immersion system; sections are examined in water or glycerin or mounted in Canada balsam through alcohol and xylol. Nuclei may be counterstained with carmine. According to Graff's method, sections are immersed for 10-15 minutes in a mixture of equal parts of a 1% aqueous solution of α-naphthol and a 0.2% aqueous solution of dimethylparaphenylenediamine. After rinsing the sections in water, they are placed for 2-3 minutes in Lugol's iodine solution. They are washed in water to which 5 drops of a saturated lithium carbonate solution are added per 10 cm³. Afterwards, the nuclei are counterstained with alum carmine, the sections are washed, and they are mounted in glycerin-gelatin. In the Nadi reactions, the presence of oxidase is revealed by the formation of indophenol blue in the form of dark blue granules (brownish from iodine in Graff's method). Reagents for the Nadi reaction are unstable; they must be kept in dark bottles and frequently renewed. The reaction for peroxidase is performed using benzidine. 1 g of benzidine is dissolved in 100 cm³ of tap water with shaking; a small amount (1-2 drops) of commercial hydrogen peroxide is added to this solution upon use. Smears and sections are kept in this mixture for several minutes, after which they are washed in water, and the nuclei are counterstained with hematoxylin or Giemsa stain; after washing, the smears are dried, and the sections are mounted in balsam through alcohol and xylol. Peroxidase granules are yellowish-brown. It should be noted that for practical purposes, the Nadi reaction for oxidase and the benzidine reaction for peroxidase can be considered equivalent, since in tissue elements upon the breakdown of phenols under the influence of hydrogen peroxide, products are formed that are identical to those yielded by oxidase reactions. A. Abrikosov.