Microsomes, Drug Oxidations and Chemical Carcinogenesis V1

P-450

ENZYMATIC ACTIVATION OF MOLECULAR OXYGEN

O. Hayaishi, O. Takikawa, M. Sono and R. Yoshida,     Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

Publisher Summary

This chapter discusses enzymatic activation of molecular oxygen. The chapter discusses about tryptophan 2,3-dioxygenase or pyrrolase, which catalyzes a rupture of the pyrrole ring of tryptophan, forming formylkynurenine as the reaction product. Tryptophan dioxygenase is a hemoprotein containing protoporphyrin IX as its sole prosthetic group. The native, ferric form of the enzyme exhibits a spectrum typical of a high-spin hemoprotein. It can be reduced to a ferrous form, which, in the presence of the substrate tryptophan, binds with oxygen to form a new spectral species with peaks at 418, 545, and 576 nm. This new spectral species, which can also be observed during the steady state of the reaction, represents a ternary complex of enzyme–substrate and oxygen and was established to be the obligatory intermediate of reaction by kinetic analysis using a stopped-flow apparatus. The chapter presents a comparison of monooxygenase activity of various hemoproteins. Turnover number of various hemoproteins is expressed in terms of mol p-aminophenol produced/min/mole of heme at 37°. The chapter also illustrates the dependency of the indoleamine dioxygenase catalyzed monooxygenase reaction.

Studies on the mechanisms of biological oxidation processes were initiated by the French chemist Lavoisier some two hundred years ago. He demonstrated that respiration is a slow combustion of various nutrients in the body, and that the inhaled oxygen oxidized carbon atoms of sugars and fats to carbon dioxide, CO2 and hydrogen atoms to water, H2O. Accordingly, Lavoisier defined the term oxidation as the addition of oxygen atoms to a substrate X, while the opposite process, that of reduction, was considered to be the removal of oxygen from an oxide, XO2 to X and O2. In the century that followed Lavoisier’s discoveries, there were many modifications and extensions of his basic ideas, and a number of theories were proposed to explain how molecular oxygen is activated and how it reacts with organic substrates.

At the onset of the present century, however, the involvement of molecular oxygen per se in biological oxidation processes was vigorously challenged by Professor Heinrich Wieland, a distinguished chemist and a Nobel Laureate from Germany. In 1923, Wieland authored a book titled, On the Mechanism of Oxidation and proposed ‘the theory of enzymatic dehydrogenation’ as the essential principle of biological oxidation (1)

According to the dehydrogenation theory, the principle of biological oxidation processes is the removal of hydrogen atoms, or more precisely speaking, electrons, from the substrate molecule XH2, and their transfer to an appropriate acceptor A, such as coenzymes and various dyes. The opposite process, namely the addition of hydrogen atoms, was defined as reduction.

What then is the role of molecular oxygen in biological oxidation processes? Oxygen molecules may, in some instances, serve as the immediate electron acceptor as shown below.

Molecular oxygen accepts hydrogen atoms and is reduced to water or hydrogen peroxide. However, it is never incorporated into the substrate as was originally proposed by Lavoisier and his contemporaries. The dehydrogenases that utilize molecular oxygen as a hydrogen acceptor are termed oxidases; cytochrome oxidase and D-amino acid oxidase are typical examples of these oxidases, each producing water and hydrogen peroxide from molecular oxygen, respectively. Thus, according to Professor Wieland, the direct addition of molecular oxygen to a substrate was considered completely irrelevant to biological oxidation.

In 1949, during a study of tryptophan metabolism in a bacterial species Pseudomonad, we isolated and purified a novel enzyme, pyrocatechase from the microorganism. Pyrocatechase catalyzed the oxidative cleavage of the aromatic structure of catechol, producing cis, cis-muconate as the reaction product (Fig. 1). Because of the unusual properties of this enzyme, we suspected that it might catalyze direct addition of O2 to substrate.

FIGURE 1 Pyrocatechase.

A set of experiments was performed in which a heavy oxygen isotope, oxygen-18 was used in air, in Experiment I, or in the form of water in Experiment II. The product, muconic acid, was isolated and analyzed for the ¹⁸O content using a mass spectrometer. Contrary to the then generally held belief, the results clearly established that the oxygen atoms incorporated into the product molecules were derived exclusively from molecular oxygen but not from the oxygen of the water molecule (2). If pyrocatechase were a dehydrogenase or an oxidase, the incorporated oxygen atoms should have been derived from water, according to the theory of Professor Wieland.

Concurrently and independently, Prof. Mason in Oregon demonstrated that phenolase of the mushroom, incorporated one atom of molecular oxygen into a substrate (3). These findings therefore established that oxygen fixation did indeed occur in biological systems. We therefore proposed that the enzymes responsible for these reactions be termed dioxygenase and monooxygenase, respectively. In essence, this was the revival of the old concept of Lavoisier. Since then oxygenases have been isolated from animals, plants and microorganisms and some have been highly purified and crystalized. The structure and the mechanism of action of oxygenases were also investigated in detail.

Dioxygenases are now subdivided into two classes as shown. in Table I. Typical examples of intra and intermolecular dioxygenases are pyrocatechase and α-ketoglutarate dependent dioxygenases, respectively.

TABLE I

Dioxygenase

Monooxygenases are also divided into two subgroups as shown in Table II. Internal monooxygenases do not require external reducing equivalents but the substrate itself provides electrons to reduce one atom of oxygen to water. Lysine monooxygenase is a typical example. On the other hand, external monooxygenases require various reducing agents as shown here.

TABLE II

Monooxygenase

Now the question arises whether or not the molecular oxygen is activated by these oxygenases and, if so, what is the nature of the enzymatically activated oxygen. Is the so-called active form of oxygen identical in dioxygenase and monooxygenase catalyzed reactions? Or, do these enzymes produce different forms of active oxygen? Since the original discovery of oxygenase in 1955, a number of so-called active oxygen has been postulated. These include singlet oxygen, superoxide anion, hydroperoxides, hydroxy radicals, oxene and so forth. However, attempts to demonstrate the involvement of these active oxygen species in a free form have so far been uniformly unsuccessful.

The first breakthrough to this formidable question came about some 12 years ago when we were studying the kinetic properties of tryptophan dioxygenase (4).

Tryptophan 2,3-dioxygenase or pyrrolase catalyzes a rupture of the pyrrole ring of tryptophan, forming formylkynurenine as the reaction product (Fig. 2). Tryptophan dioxygenase is a hemoprotein containing protoporphyrin IX as its sole prosthetic group. Parenthetically I might mention that there has been some question about the involvement of copper but now it is firmly established that copper is not required for its catalytic activity. In 1957, we demonstrated, with the use of oxygen-18, that molecular oxygen was directly incorporated into formylkynurenine.

FIGURE 2 Tryptophan 2,3-dioxygenase (pyrrolase).

The native, ferric form of the enzyme exhibits a spectrum typical of a high spin hemoprotein (Fig. 3). It can be reduced to a ferrous form, which, in the presence of the substrate tryptophan, binds with oxygen to form a new spectral species with peaks at 418, 545 and 576 nm. This new spectral species, which can also be observed during the steady state of the reaction, represents a ternary complex of enzyme substrate and oxygen and was established to be the so-called obligatory intermediate of reaction, by kinetic analysis, using a stopped-flow apparatus. Now, what is the nature of this spectral species?

FIGURE 3 Spectra of tryptophan 2, 3-dioxygenase.

The positions of the absorption maxima of this new spectral species are almost identical to those of oxygenated hemoproteins such as oxyhemoglobin, oxymyoglobin and peroxidase Compound III (Table III). Please note, however, that the oxygenated form of tryptophan dioxygenase was observed only in the presence of the substrate, tryptophan, while the other oxygenated forms represent binary complexes of oxygen and heme proteins.

TABLE III

Absorption maxima of oxygenated heme proteins

Following our demonstration of the oxygenated form of tryptophan dioxygenase, a similar spectral species representing presumably an oxygenated form of cytochrome P-450cam of Pseudomonas putida was reported by Ishimura, Ullrich and Peterson in Dr. Estabrook’s laboratory (5) and also by Dr. Gunsalus and associates. Furthermore, similar oxygenated forms of enzymes were reported with flavoprotein monooxygenases by Massay, and with a non-heme iron dioxygenase by Fujisawa and coworkers.

(Fig. 4). However, it is distinctly different from the classical hepatic tryptophan dioxygenase.

FIGURE 4 Indoleamine 2, 3-dioxygenase.

Tryptophan dioxygenase (TPO) has been found only in the liver, while indoleamine dioxygenase (IDO) is ubiquitously distributed in various organs and tissues. TPO is strictly specific and acts only on tryptophan, whereas IDO exhibits a broad substrate specificity and catalyzes the indole ring cleavage of not only tryptophan but also 5-hydroxytryptophan, tryptamine, serotonin, and so forth. Lastly, TPO utilizes molecular oxygen while IDO requires and utilizes the superoxide anion, a univalently reduced molecular oxygen, for activity. Hence it may be termed superoxygenase. The evidence for the participation of the superoxide anion in the IDO-catalyzed reaction has been published in detail (6) and is summarized below.

First, the superoxide anion is required and utilized by the enzyme. The superoxide anion can be supplied as relatively stable potassium salt, KO2, or generated either chemically or enzymatically. The superoxide is required for the initiation of the reaction as well as for maintaining the steady state of the reaction. Second, scavengers for the superoxide anion such as superoxide dismutase, Tyron and so forth, inhibit the reaction but scavengers for singlet oxygen of hydroxy radicals show no effect whatsoever, even at high concentrations. Third, a heavy oxygen isotope, oxygen-18, is incorporated into the product of the reaction from K¹⁸ O2. However, when the reaction is carried out in the presence of heavy oxygen containing air, oxygen-18 is also incorporated into the product of the reaction albeit to a lesser extent. In addition, several lines of evidence indicates that O−2 is utilized by this enzyme in vivo and is the rate limiting factor under physiological conditions. These results, taken together, support the conclusion that, indoleamine dioxygenase is a unique oxygenase that requires and utilizes the superoxide anion and decomposes indoleamine derivatives.

When the superoxide anion was mixed with the ferric form of enzyme, a new spectral species was immediately observed (Fig. 5). When molecular oxygen was introduced into a cuvette containing the ferrous form of enzyme, an exactly identical spectral species was observed, indicating that this new spectral species represents the heme-oxygen complex. The spectral properties of this species is essentially identical to properties of so-called oxygenated hemoproteins such as oxyhemoglobin, oxymyoglobin, peroxidase Compound III and the ternary complex of tryptophan dioxygenase•substrate•oxygen (Table III). Although this binary heme-dioxygen complex is relatively stable and the half life is approximately 20 minutes at pH 7.0 and 24°, it decomposes instantaneously upon addition of the substrate,