Peroxidase

13 Peroxidase ISAO YAMAZAKI I. Introduction II. Functions of Peroxidases A. Higher Oxidation States of H R P Β. IAA Oxidation III. Structure of Peroxidases A. Chemical Structure B. Electronic Structure of the Heme IV. Relationship between Structure and Function A. Peroxidases with Unnatural Heme Groups B. Natural Peroxidases with Heme Groups Different from Protoheme C. Comparison of the Function of H R P with Other Hemoproteins V. Conclusion References

.

535 536 536 539 545 545 546 547 548 550 551 553 554

I. I N T R O D U C T I O N * Peroxidases are enzymes, whose primary function is to oxidize molecules at the expense of hydrogen peroxide. Probably because of their wide dis­ tribution, especially in plants, and their dramatic catalysis of colored product formation, these enzymes have been among the most extensively investigated since the beginnings of enzymology. A detailed historical review of these enzymes was given by Saunders et al. (1). The names "peroxidase" and "oxygenase" are analogous because of their strong specificities for peroxide and oxygen, respectively. From the point of view of function, however, peroxidase is rather similar to oxidase, the name of which is now used in a narrow sense for electron transfer oxidases. The name "oxygenase" is restricted to enzymes that catalyze the incorpora* Abbreviations; HRP, horseradish peroxidase; CCP, cytochrome c peroxidase; TP, tryptophan pyrrolase; IAA, indole-3-acetie acid; D H F , dihydroxyfumaric acid; N A D H , reduced form of nicotinamide-adenine dinucleotide; and ESR, electron spin resonance. 535

536

ISAO YAMAZAKI

tion of atmospheric oxygen into substrate molecules (2). Based on the acceptor specificity and by analysis of the final reaction products, the distinctions between electron transfer oxidase, oxygenase, and peroxidase appear to be established. Reactions of mixed types such as monooxygenase (mixed function oxidase by Mason's terminology, refs. 3 and 4) and peroxidase-oxidase have been reported. If peroxide is an intermediate product of 0 2 reduction it might be said that in many cases peroxide metabolism is involved as a part of the overall oxygen metabolism. Detailed analyses of the mecha­ nism of 0 2 metabolism have revealed that three types of reactions are correlated in complicated ways. oxidase (electron transfer) oxygenase ^ p e r o x i d a s e

The recently established idea of oxyferroperoxidase structure for so-called Compound III would make it easy to relate the function of peroxidase with those of electron transfer oxidase and oxygenase. II. FUNCTIONS O F PEROXIDASES Besides peroxidatic oxidation of electron donor molecules various re­ actions have been found to be catalyzed by peroxidase. These are aerobic oxidations of D H F (5, 6), IAA (7-9), triose reductone (10), NADH (11, 12) and naphtohydroquinone (13, 14), hydroxylation of aromatic molecules (15, 16), formation of ethylene from methional (17), halogenation (18, 19), and antimicrobial activity (20, 21). The common feature of these reactions appears to be an involvement of H 2 0 2 . Thus, it is likely that their mecha­ nisms are controlled by a unique feature of the peroxidase reaction. A . Higher Oxidation States of HRP /. HRP Compounds

Formed in the Catalytic

Reaction

Peroxidase catalysis is characterized by the one-electron oxidation of donor molecules. The following reactions have been confirmed (22-24): Peroxidase + H 20 2 —• Compound I Compound I -f AH 2 —• Compound II + Α Η · Compound II + AH 2 —> peroxidase + AH2 AH- -> A + A H 2 (or AH-AH)

(1) (2) (3) (4)

537

13. PEROXIDASE

Compounds I and II are thus found to be obligatory enzyme intermediates in the overall peroxidase reaction. Judged by the hyperfine structure of ESR spectra, the free radicals derived from several donor molecules in the above reactions were considered to be free in solution (24, 25). These free radicals are very reactive, and their reactions will result in a variety of peroxidase functions. During aerobic oxidations of D H F and NADH catalyzed by H R P the enzyme was converted into an another compound, called Compound I I I (5, 6, 12). The compound had been identified as a product formed in the presence of excess H 2 0 2 (26, 27). 2. Formation

of HRP Compound

HI

An oxyferroperoxidase structure has been suggested for peroxidase Compound III (3, 28-31). This idea, however, was not generally ac­ cepted since reduced peroxidase was thought to be oxidized to the ferric enzyme without an intermediate similar to Compound I I I (32-34). Re­ cently, the oxyferroperoxidase structure of Compound III has been con­ firmed by the following experiments, (a) Ferroperoxidase did react with oxygen to form Compound III when excess hydrosulfite that reduced Compound III was not present (35-37). (b) Compound I I I was formed by photolysis of an aerobic solution of CO-ferroperoxidase (38, 39). (c) Titrimetric experiments showed that Compound I I I was at a threeequivalent oxidized state above the ferric enzyme (37, 39, 40). It was shown by Chance (41) and George (27) that Compound I I I was formed from the reaction of Compound II and H 2 0 2 . One more path of Compound I I I formation via reaction between the ferric enzyme and superoxide anion was suggested by Yamazaki and Piette (30). The latter reaction has been considered to be involved in the formation of Compound III during aerobic oxidations of D H F (30, 31) and NADH (12) catalyzed by peroxidase. Participation of superoxide anions in the above reactions was finally confirmed in the cases of H R P (42) and myeloperoxidase (43, 44) using superoxide dismutase (45) which accelerates the decomposi­ tion of superoxide anions. 3. Reactions

of Higher Oxidation Forms of HRP

Figure 1 shows the relationship between five redox forms of HRP. All these forms could be obtained in fairly stable states under suitable experi­ mental conditions and have been crystallized using a basic H R P prepara­ tion, named "isoenzyme F " (46). Higher oxidation forms of H R P could be reduced to the ferric enzyme by various electron donors. The most active form is Compound I. Chance and his colleague showed that nitrous acid

538

ISAO YAMAZAKI Ferric form 3

Ferrous form

4 Compound Π

6-

Compound ΙΠ (oxy form)

*-5

Compound I

FIG. 1. Five redox states of HRP. The numbers formally indicate the effective k k oxidation number of the peroxidase heme: 2 + 0 2 ~^ 6, 2 + H 20 2 ~ 4, 3 -f- H 0 2 — 6, 3 + H_ 20A2 —" 5, and 4 + H 20 2 —* 6; others are one-electron steps. Reaction paths, 3 ^ 5 4 ^ 3 indicate a catalytic cycle of the enzyme.

(47) and p-aminobenzoic acid (48) reduced Compound I 100 and 25 times faster than Compound II. Similar results were obtained by Cormier and Prichard (49) with luminol, by Dunford and colleagues with ferrocyanide (50) and iodide (51, 52), and by Yamazaki et al. (53) with ascorbic acid and anthranilic acid. The conversion of H R P Compound I to the ferric enzyme without an appreciable formation of Compound II was reported by Bjorksten (54) and Roman and Dunford (52) using iodide as an electron donor. Horseradish peroxidase Compound III also reacted with various electron donors which are not autoxidizable (31, 39, 55). Compound III was less reactive with electron donors than Compound II, while in the absence of such donors Compound III was less stable than Compound II. The sta­ bility of Compound II varied greatly with enzyme preparations, but that of Compound III was almost independent of the purity of enzyme prepara­ tions (39). Horseradish peroxidase Compound III underwent a spontaneous decay to the ferric enzyme without detectable intermediates like Compounds I and II (36, 37, 39). This might be explained by assuming that the oxidative decomposition of Compound III occurs in the presence of one-electron oxidants such as Compounds I and II. Compound III + e~ —* peroxidase + 0 2

(5)

Rate constants of the reactions of Compound III with Compounds I and

539

13. PEROXIDASE

II could be measured (39). However, the release of 0 2 during the reaction has not been confirmed. For the decomposition of Compound III a mecha­ nism in which dissociation of Compound III into Compound II and H 2 0 2 is rate limiting was proposed (39), but this needs further proof. The reaction of Compound III with electron donors would be of par­ ticular interest. Undoubtedly, 0 2 is activated when it combines with ferroperoxidase. It might be reasonable to assume that Compound III is re­ duced by electron donors via the intermediates Compounds I and II. As mentioned above, these intermediates are much more active oxidants for the electron donors so far used. Therefore, an ingenious device would be needed to identify the intermediates in the reduction process of Compound III. 4. Oxidation-Reduction

Potentials

Harbury (33) measured the oxidation-reduction potential of H R P for the reaction, ferric H R P ferrous HRP. The potential was —0.271 at pH 7.0. For the reaction, ferric H R P H R P Compound II, the reversible potential has not been obtained. George (56, 57) showed that H R P could be oxidized by a variety of one- and two-electron oxidants to compounds similar to Compounds I and II. Fergusson (58), however, suggested a possibility that the formation of such compounds occurred via the inter­ mediate formation of H 2 0 2 . Observing that H R P Compound II was re­ duced by chloroiridite ions (1.02 V) but not by ferrous tris-2,2'-dipyridyl and tris-o-phenanthroline ions (1.06 and 1.14 V), George (22) proposed a tentative value of about 1.0 V for the oxidation-reduction potential of the Compound II and ferric H R P couple. He also suggested that Compound I could be a more powerful oxidizing agent than Compound II by about 0.3-0.6 V. It might be generally said that the reactions shown in Fig. 1 tend to proceed almost to completion under physiological conditions, and it is difficult to measure these equilibrium constants with the usual methods except for the ferrous and ferric couple. The direct conversion from ferrous H R P to Compound II was demonstrated by Noble and Gibson (59). B. I A A Oxidation J . General Mechanism

of Peroxidase-Oxidase

Reaction

Among numerous substrates of peroxidase-oxidase reactions the IAA reaction has been most intensively studied. The principal feature of the peroxidase-oxidase reaction appeared to be the same irrespective of sub­ strate molecules. The earlier studies have been reviewed by Nicholls (60).

540

ISAO YAMAZAKI

The mechanism of Fig. 2 was proposed by Yamazaki and Piette (30). The common properties of the reaction are (a) a catalytic amount of H 2 0 2 is necessary to initiate the reaction by forming the free radicals of donor molecules (catalase inhibits the reaction); (b) redogenic molecules (60, 61) such as ascorbate inhibit the reaction by consuming H 2 0 2 or by inter­ action with active intermediates having oxidizing equivalents (61); (c) oxidogenic molecules (60, 61) such as phenols promote the reaction by in­ creasing the rate of free radical formation of substrate molecules (61); and 2 (d) Mn + ions promote the reaction, probably through interaction with superoxide radical species or with free radicals of oxidogenic molecules. The extent of activation or inhibition caused by a given molecule varies greatly with substrates and experimental conditions. Although this mecha­ nism appeared to be essentially valid for the reaction with IAA, evidence has been accumulated which indicates that the reaction of H R P with this substrate differs from those with other substrates such as NADH and DHF. 2. Problems

in IAA

Oxidation

It is very interesting to note here a feature of IAA-HRP reactions that is rather similar to an oxygenase type. Except for IAA the oxidation prodRH

Compound m

1/2 (H 20 2 + Oa)

FIG. 2. General mechanism of peroxidase-oxidase reaction. X H is an oxidogenic molecule which promotes the reaction. The mechanism will vary with RH molecules depending upon the reactivity with Compound III, the stability of a product, ROO« 2+ 3+ radical, etc. F e p and F e p denote ferrous and ferric HRP, respectively.

541

13. PEROXIDASE

ucts of peroxidase-oxidase reactions are principally two-electron oxidized forms. By the efforts of many workers (7,62-66) the following stoichiometry has been confirmed:

(6)

Η

Η

The products were found to be indole-3-aldehyde and 3-methylene oxindole. Morita et al (64) showed that the dominant product was indole-3aldehyde when higher enzyme concentrations were used. Apparently the reaction is similar to the lactate oxidative decarboxylase reaction, which is a monooxygenase type (67) or an internal mixed function oxidase type (4). Indole-3-acetic acid was found to react with H R P Compound I I I at a relatively high rate (31, 39, 55). In this respect the reaction is similar to the tryptophan pyrrolase reaction, in which the oxygenated enzyme was considered to be an obligatory intermediate (68, 69). Many features of the mechanism of the IAA reaction, however, still remain to be elucidated. r The key points of the mechanism w ould be summarized in the following way. a. The IAA free radical formed by peroxidase catalysis is an important intermediate (9, 65, 66, 70-74). Hinman and Lang (65) proposed a mecha­ nism in which the radical reacts with molecular oxygen to form 3-methylene oxindole as a main product through several nonenzymic steps. Though it is unknown whether the radical is free or attached to the enzyme, it may react with external oxidants such as ferric cytochrome c and ferric o-phenan­ throline complexes (70). A question which arises is why the nature of the final products depends upon the concentration of enzymes in the reaction (64-66). b. During the reaction with IAA a part of the enzyme is observed as an intermediate, spectrally similar to Compounds II and I I I (66, 70, 73-77). From the visible spectrum between 500 and 600 nm the inter­ mediate is judged to be Compound III at around pH 4.0 (66) and Com­ pound II at pH 5.0 (75, 78). It has been concluded that Compound III

542

ISAO YAMAZAKI

accumulated during the reactions of D H F and NADH is not a Michaelis type of intermediate (6, 12, 30). Since IAA reacts with H R P Compound III at a significantly high rate, the formation of Compound III during the IAA oxidation must have a positive meaning in the catalysis. However, it is still uncertain whether or not the main reaction proceeds via Com­ pound II or III, mostly because of the difficulty in distinguishing between these two H R P compounds. c. A small amount of H 2 0 2 eliminates the lag phase and promotes the 0 2-consuming oxidation of IAA under certain experimental conditions (7, 66, 70-72, 79). It is also true that the inhibition by catalase is negligible under certain experimental conditions (75, 78). Consequently, the role of H 2 0 2 does not appear to be essential even as an initiator of the reaction. Using superoxide dismutase it has recently been found that superoxide anions are not involved in the reaction of IAA with H R P (77, 78). This fact seems to be a peculiar property of the IAA reaction since the oxida­ tions of NADH and D H F by H R P are strongly inhibited by superoxide dismutase (78). This fact is compatible with the mechanism (65, 72) that the IAA peroxide radical instead of the superoxide anion radical is a product of the reaction between the IAA radical and 0 2 . Questions to be answered are how the IAA radical can be formed in the abesnce of H 2 0 2 and how H R P Compound III is formed without an involvement of super­ oxide anions. If the absolute necessity for H 2 0 2 in the activation process does not apply to the IAA reaction, the mechanism would be different from the general peroxidase—oxidase reaction shown in Fig. 2. Fox et al. (75) have proposed a mechanism which involves a rapid equilibrium exchange of biradical molecular oxygen with water through a coordination position of the ferric iron to yield an oxygenated form of HRP. They suggested that oxygenated H R P attacks IAA to form an intermediate compound similar to Compound I. Though the mechanism involving an activation process without H 2 0 2 participation is not necessarily supported by un­ ambiguous evidence, it is suggestive. On the other hand, Ricard and Nari (73) showed that H R P was reduced directly by IAA under anaerobic conditions at acidic pH. This reduction seems to be too slow to explain the rapid oxidation of IAA (78) but is in accord with the known properties of HRP. Since interactions between H R P and donor molecules have been demonstrated (53, 80), it would be reasonable to assume an equilibrium between H R P and IAA as the first step of the reaction. The rapid oxidation of IAA accompanied by the formation of H R P peroxide compounds after the addition of IAA would be tentatively explained by a mixed mechanism

543

13. PEROXIDASE

/ RH

ι R-

FIG. 3. Mechanism of I A A ( R H ) oxidation (78). Solid lines indicate maintenance reactions. A radical chain process may be involved, with propagation steps which can be generalized as R-

+ 0 2- * R O O .

ROO- + RH ^

ROOH +

R-

as shown in Fig. 3. In this mechanism it is assumed that IAA peroxide (ROOH) and its free radical (ROO-) serve as H 2 0 2 and Η 0 2 · , respectively, in the general peroxidase-oxidase reaction of Fig. 2. According to Yonetani et al. (81), CCP can react with relatively large peroxide such as 2,5dimethylhexane 2,5-dihydroperoxide to form its peroxide compound. Schonbaum (82) has shown that reaction of H R P with m-nitroperoxybenzoate yields Compound I with the simultaneous release of m-nitrobenzoate. The mechanism proposed by Ricard and Nari (73, 74) might be involved to some extent in the reaction at acidic pH. 3. Heme Degradation

during IAA

Oxidation

Yokota and Yamazaki (55, 83) reported that during the oxidation of IAA, H R P was converted into a compound similar to Compound IV which was found by Chance (84) in the presence of excess methyl peroxide. This fact appears to be in accord with the observation of Fox et al. (75) that a considerable amount of H R P is inactivated during the oxidation of IAA. Further studies (85) revealed that one mole of CO is released for one mole of H R P transformed and the product of H R P is similar to choleglobin (86), details of which have been reviewed by Lemberg (87) and O'hEocha

544

ISAO YAMAZAKI

(88). Since the efficiency of the transformation was higher when IAA re­ acted with Compound I than Compound II or I I I (78), a tentative scheme may be proposed as shown in Fig. 4. This transformation was specific for the isoenzymes of H R P and was observed in the case of isoenzymes H R P Β and C but not H R P A and F. Bagger and Williams (89) observed a compound with an intense ab­ sorption band in the near infrared when excess H2O2 was added to H R P . They suggested that this compound, called P-940, is an intermediate in the conversion of H R P into P-670 caused by excess H 2 0 2 ; P-940 was also observed as an intermediate in the reaction of H R P with IAA (78).

Compound I 4+ Fe

Compound Π 4+ Fe

Fe

Verdoheme

3+

Oxyheme

Η

Η

Indole-3-aldehyde

3-Methylene oxindoli

FIG. 4. Heme transformation in the reaction with I A A . The mechanism is based on the following observations (78): (1) During the catalytic oxidation of I A A the dis­ appearance of Compound I I and the appearance of verdoheme were observed at the same time; (2) in the stoichiometric reaction (not catalytic) the heme transformation was most effective when I A A was added to Compound I. The latter half mechanism is from Lemberg (87).

545

13. PEROXIDASE

III. STRUCTURE O F PEROXIDASES A . Chemical Structure 1. Prosthetic

Group

The prosthetic groups of HRP, Japanese radish peroxidase, CCP, and chloroperoxidase are known to be ferriprotoporphyrin IX. The heme groups in animal peroxidases are much more tightly bound to the protein than are the hemes in plant peroxidases, and their chemical structures have not been thoroughly clarified. Thyroid peroxidase has a heme group which is not covalently linked to the apoenzyme and is closely related to protoheme (90-92). The prosthetic group of milk peroxidase is bound by covalent bonds to the protein and can be separated from the protein by reductive cleavage with HI. The isolated heme gave rise to mesoporphyrin IX, and it was suggested that the heme in milk peroxidase is directly re­ lated to protoheme (93). The spectral properties of myeloperoixdase are quite different from other peroxidases. It was reported that the pyridine hemochromogen spectrum was similar to that of heme a (94, 95). 2.

Protein

Little is known about the chemical structure of peroxidases. Amino acid analyses were carried out for five isoenzyme preparations of H R P (96) and for two isoenzyme preparations of Japanese radish peroxidase (97, 98). The conformation of two H R P isoenzymes Ai and C was investigated by means of circular dichroism, and it was suggested that the active sites were similar in both isoenzymes but that small differences did exist (99). The environment surrounding the heme of CCP was found to be hydrophobic (100). This might be the case with all plant peroxidases. The hydrophobic structure of the heme environment of myoglobin and hemoglobin has been disclosed by X-ray analyses. A possibility that the fifth coordination position in H R P is occupied by an imidazole group of the protein was suggested by several workers 14 (101-104). By elaborate experiments using NO, Yonetani (104) con­ cluded that the fifth ligand of the heme iron in CCP and H R P may be the imidazole group of a histidine residue and that the unpaired electron of NO sits in the sixth coordination position although it is considerably delocalized to the heme iron and the proximal nitrogen in these NO com­ pounds of ferrous peroxidases.

546

ISAO YAMAZAKI

Β. Electronic Structure of the Heme The electronic structure of the heme iron has been a subject of con­ siderable interest. Most of the earlier studies were carried out by TheorelPs group (32, 105) using the technique of magnetic susceptibility. Theorell and Ehrenberg (106) measured the number of odd electrons present in some peroxide compounds of myoglobin, peroxidase, and catalase. Electron spin resonance spectroscopy was also successfully used for peroxidases in frozen solutions (107, 108). The major advance, however, has recently come from the work of Yonetani and his colleagues. Through a series of elegant ESR studies, mostly on CCP, they were able to measure the electronic structure of the heme iron in various states of the enzyme (109, 110). 1. High Spin and Low Spin States of Ferric

Peroxidases

The spin state of the ferric heme iron can be investigated by means of paramagnetic susceptibility and optical, ESR, and Mossbauer spec­ troscopies. The measurements of paramagnetic susceptibility of CCP (111, 112) and H R P (113, 114) gave precise information on a thermal equilibrium between high and low spin states. The information on the anisotropy of paramagnetic susceptibility in the heme plane can be pro­ vided by ESR spectroscopy (109, 115, 116). 2. Ferryl Structure

in Compounds

I and II

On the basis of the oxidation equivalent and the comparison with model 2+ systems, George (27) suggested a ferryl ion, F e 0 , as a possible structure of the iron atom of Compound II. The magnetic susceptibility data (106) which indicated the existence of two unpaired electrons is consistent with this idea. The conclusive support for the idea was given by means of Mossbauer spectroscopy (117-120). The electronic structure of Compound I had been a complete mystery until Mossbauer spectroscopic data showed that the electronic structure of the heme iron was the same in Compounds I and II (117, 119, 120). One of the two oxidizing equivalents in Compound I was thus accounted for by the loss of an electron from the iron atom. Using metal-porphyrin com­ plexes, Dolphin et al. (121) showed that the optical absorption spectra of Compounds I of catalase and H R P were similar to those of π-cation radicals of porphyrin. They concluded, therefore, that the second oxidizing equivalent in Compound I originates in the 7r-cation radical of the porphy­ rin. The failure to observe an ESR signal arising from the radical of Com­ pound I was explained by an exchange interaction of a single electron localized on the porphyrin ring with the spin of electrons localized on the iron.

13.

547

PEROXIDASE

Yonetani et al. (122) clearly observed an ESR signal of a free radical in the primary peroxide compound of CCP. This compound possesses two oxidizing equivalents above the ferric enzyme but gives an optical spectrum similar to those of Compounds II of catalase and HRP. In the primary peroxide compound of CCP the second oxidizing equivalent might be localized in an amino acid residue of the protein. The formation of a small amount of free radical was also observed in the reaction of H R P with H 2 0 2 (108). 3. Compound

III

(Oxyperoxidase)

The electronic structure of the heme of oxyhemoproteins has been dis­ cussed for a long time, mostly with hemoglobin and myoglobin. Oxyhemo­ globin is known to be diamagnetic (123). However, it has been suggested by several workers (124-126) that the migration of an electron occurs from the heme iron to the oxygen. As discussed by Peisach et al. (126), two unpaired electrons are not necessarily localized in the iron and oxygen atoms and the spins could combine to cancel their paramagnetism. It would seem difficult to confirm the migration of an electron from the iron to the oxygen by Mossbauer spectroscopy. The oxygen molecules in oxy-TP and H R P Compound III are considered to be more activated than those in oxyhemo­ globin and oxymyoglobin, but at present no effective physical technique has been reported to distinguish the electronic structure of these hemoproteins.

IV. RELATIONSHIP BETWEEN STRUCTURE A N D

FUNCTION

Obviously, the information on the chemical structure of peroxidase is quite insufficient to discuss the relationship between the structure and function. There are a number of protein groups which contain common prosthetic atoms or molecules such as iron, copper, flavin, heme, and pyridoxal. New functions of these groups acquired by incorporation into specific environments of proteins have attracted considerable attention. Of these, the heme group seems to be one of the most extensively inves­ tigated molecules because of its biological importance and its characteristic properties which can be measured by various physical and chemical meth­ ods. Most hemoproteins contain protoheme as the prosthetic group. Peroxi­ dases of plants contain protoheme while those of animals contain heme more or less different from protoheme. Accordingly, it might be supposed that the function of hemoproteins is not strongly influenced by modifica­ tions in the heme moiety.

548

ISAO YAMAZAKI

A . Peroxidases with Unnatural Heme Groups Since Theorell (127) found that H R P was reversively split into apo-HRP and protoheme, a number of experiments have been carried out in order to clarify the effect of heme modification on the functions of hemoproteins. Artificial hemoproteins with unnatural hemes have been prepared for hemoglobin (128), myoglobin (129), H R P (132-136), CCP (137, 138), and cytochrome b 5 (139). These results are summarized in Fig. 5. It may be concluded from the results that substituents at positions 2 and 4 of deuteroheme have no significant effect on the functions of hemo­ proteins. Phillips (140) showed that the electron-withdrawing capacities of 2,4 substituents of deuteroporphyrin are correlated with properties such as spectroscopic behavior of the porphyrin and basicity of the pyrrole

A r t i f i c i a l h e m o p r o t e i n s with unnatural h e m e

Natural h e m o p r o t e i n s H e m e different from p r o t o h e m e

Protoheme

Function i s r e t a i n e d

Function is lost

(a)

(b) 0 2 Carrier

Hemoglobin

Dimethyl proto-, dimethyl meso-, etio-, meso-, d e u t e r o - , h e m a t o - , (128)

Myoglobin

D e u t e r o - , m e s o - , (129)

I Oxygenase

- Tryptophan p y r r o l a s e

P-450

I I

3+

Catalase

Compound I — « - F e

(Chloroperoxidase) Compound I -Compound Π

**

Μ .Ι ο, (Myelo -| per.)

(d) Milk per.

(e)

(Thyroid per.)

Plant peroxidase

Hemato-, m e s o - , deutero-, (132,133,134,135), diacetyl deutero-, (135,136),monom e t h y l p r o t o - , (134)

Cytochrome c peroxidase

Hemato-, meso-, deutero-, (137), e t i o - , dialkyl p r o t o (g), (138)

(f)

I

Electron transfer oxidase Electron carrier

(h) Cyt.d-

Cyt. oxidase

(c)

( Cyt.b

Dimethyl proto-, (134,135)

) Cyt. c H e m a t o - , m e s o - , d e u t e r o Cyt. b 5 (139)

FIG. 5. Hemes and functions of hemoproteins. Parentheses imply that the position is still in question, (a) Left and right sides indicate red and blue shifts in absorption max­ ima, respectively, (b) Left side of the dotted line means a possible involvement of a structure of chlorine type or other unusual types, (c) Chloroperoxidase has strong catalase-type functions (130), and higher oxidation states other than Compound I have not been identified (131). (d) Cytochrome oxidase-like derivative of myeloperoxidase formed by mild acid treatment (see text), (e) Salivary peroxidase is found to be the same as milk peroxidase (141), and intestinal peroxidase is also very similar to milk peroxi­ dase (144). (f) Higher oxidation states have not been observed, (g) The reaction with ferrocytochrome c is lost, (h) A chlorine type.

549

13. PEROXIDASE

nitrogen. The basicity has been measured in terms of ρϋΓ3, defined as +

p # 3 = p H - l o g ( P H 2) / ( P H 3 )

(7)

where P H 2 and PH 3+ are neutral and monocationic species of the porphyrin, respectively. Makino and Yamazaki (135) found that the basicity of the pyrrole nitrogen is reflected directly in the basicity of the heme iron in HRP, which can be expressed in terms of the proton dissociation constant, ΚΛ p # a = p H - log(alkaline H R P ) / ( a c i d H R P )

(8)

Although the value of p2fa varied with different isoenzyme preparations of HRP, the dependence of ρΚΛ of each isoenzyme preparation upon 2,4 substituents was identical as can be seen in Fig. 6. For given 2,4 substituents the following equations were given to the relation between pjff3 of the porphyrin and p K a of H R P containing the heme: P # a ( A ) = p # 3 + 4.4

For H R P - A , For H R P - ( B + C),

ρΚΛ(Β

+ C) = p #

3

+ 6.0

(9) (10)

The difference of 1.6 between two constants in the above equations may be attributable not only to the difference of the local charge distribution of the protein but also to the specific amino acid residue which might be responsible for the proton dissociation reaction. Thus, artificial H R P con­ taining a 2,4-diacetyldeuterohemin group had ρΚΛ values much different

p K Q of H R P - A FIG. 6. Correlation of dependence of heme substitution upon p K a (135). ( O ) Cor­ relation between H R P - A and H R P - ( B + C) and ( # ) pK3 values (140) plotted against ρΚΛ of H R P - A .

550

ISAO YAMAZAKI

from that of natural HRP. The stability of Compounds II and I I I of 2,4-diacetyldeutero-HRP increased, while that of Compound I decreased as compared with natural H R P (135). B. Natural Peroxidases with Heme Groups Different from Protoheme It is of special interest to note that peroxidases so far isolated from animal tissues contain prosthetic groups different from protoheme. The common feature of animal peroxidases is that the heme is tightly bound to the protein. Except for thyroid peroxidose (92) the binding is considered to be a covalent type. By immunodiffusion analysis salivary peroxidase was found to be identical with milk peroxidase (141, 142). Eosinophil peroxidase (143) and intestinal peroxidase (144) had spectroscopic proper­ ties very similar to milk peroxidase. Morell and Clezy (145) concluded that the heme in milk peroxidase contains a strongly electrophilic substituent, conjugated to the porphyrin ring, which is labile to strong alkali. Judging from the spectral property the electronic structure of the heme in milk peroxidase appears to be similar to that of 2,4-diacetyldeutero-HRP. In this connection it should be noted that Compound III of milk peroxidase is as stable as Compound III of 2,4-diacetyldeutero-HRP and the decay time at 2° might be more than a day (40, 135). Of the known peroxidases myeloperoxidase has unique spectral proper­ ties quite different from other peroxidases. The possibility that myelo­ peroxidase contains a heme group similar to heme a was suggested by Schultz and Shmukler (94) and Newton et al. (95). Odajima and Yamazaki (146) found that by acid treatment myeloperoxidase of normal pig leukocytes was converted to a derivative still possessing peroxidase activity (60% of the original) but having absorption spectra quite similar to cytochrome oxidase. The spectral comparison was applied to their respective forms such as ferric, ferrous, cyanide-ferric, and CO-ferrous. Like cytochrome oxidase myeloperoxidase probably contains two iron atoms per molecule (147150) which have different reactivities with H 2 0 2 (151) and cyanide (150). It was concluded (146) that the electronic structure of the heme in myelo­ peroxidase is distorted by interactions with the protein and the distortion can be removed by mild modification of the protein structure to form a spectrally usual type of hemoprotein containing heme a analogues. The mechanism of the conversion might be analogous to that of the conversion from P-450 to P-420 (152, 153); for instance, on modification the absorp­ tion maxima of reduced myeloperoxidase (475 and 637 nm) were shifted to shorter wavelengths by about 30 nm (146). From the appearance of these properties myeloperoxidase looks much more similar to cytochrome oxidase than HRP. The function of the myeloperoxidase heme is, how-

551

13. PEROXIDASE

ever, found to be essentially the same as that of HRP. The existence of five redox states of myeloperoxidase has been confirmed (154). C. Comparison of the Function of HRP with Other Hemoproteins In plant tissues (96, 155, 156) and in leukocytes of normal blood (157, 158) multiple forms of peroxidase have been observed. Though the amino acid composition varies greatly for the isoenzyme preparations, the basic catalytic property of these preparations described in Fig. 1 is essentially the same. On the other hand, the pattern of physiological functions of the heme is characteristic of the group of hemoproteins, and the distinction between these groups is obviously ascribed to the protein moiety (Fig. 5). Based on the known three-dimensional structures of hemoglobin and myoglobin the dependence of the catalytic properties of the heme upon the protein structure will be extensively studied in the future. Since the catalytic properties of five redox states of the heme are clarified to a con­ siderable extent in the case of H R P it might be worthwhile to compare them with those of other hemoproteins. The outline is shown in Fig. 7.

Myoglobin hemoglobin

ζζ\ ^

(4)

®

Catalase

® Cytochrome

(g\

Tryptophan / g V pyrrolase ^ ©

P-450

5

(D

•

® Cytochrome oxidase

FIG. 7. Five redox states and functions of hemoproteins. Solid circles, identified; dotted circles, still in question; =>, main function. Catalase: The details are reviewed by Nicholls and Schonbaum (159). The reaction from 5 to 2 needs sodium azide or hy­ droxyl amine. Cytochrome c peroxidase: according to Coulson et al. (160). Cytochrome oxidase: according to Okunuki (161). P-450: according to Ishimura et al. (162). Hemo­ globin, myoglobin: The addition of H 20 2 to myoglobin forms ferrylmyoglobin (163). The state 5 is stable at pH 8 and the reaction from 2 to 4 occurs by H 20 2 in the case of myo­ globin (164). Tryptophan pyrrolase: according to Ishimura et al. (69).

552

ISAO YAMAZAKI

1. Oxida tion-Reduction

Potentials

of the Ferric and Ferrous

Couple

It is known that unlike other hemoproteins native catalase cannot be reduced to the ferrous form by hydrosulfite. The oxidation-reduction potential of most hemoproteins varies from —0.27 V (HRP) to 4-0.28 V (cytochrome a) at pH 7. The potentials of hemoglobin, myoglobin, and cytochromes are between these limits. Williams (165) suggested that the potential depends on the ligand electronegativity. The electronegativity of substituents conjugated to the porphyrin ring may profoundly affect the potential. 2. Compound

II

Compound I I of myoglobin is fairly stable (163). Compound I I of cat­ alase has little catalytic activity (159). Compound I I of CCP is an obliga­ tory intermediate in the peroxidase reaction of CCP, but it is too reactive to isolate as a single species (160). 3. Compound

I

The form and stability of this oxidation state is an important criterion which indicates the dependence of the catalytic function of the heme upon the protein structure. In the reaction of ferric hemoproteins with H 2 0 2 three types are known: HRP, catalase + H 20 2 - * (ferryl + χ-cation radical in porphyrin) CCP -f- H 20 2 —• (ferryl + free radical in protein moiety) Hb, Mb -f H 20 2 —• (ferryl) + nonspecific products derived from HO

Since one oxidizing equivalent is retained in the iron atom of these hemo­ proteins, the difference between them can be ascribed to the state of a second oxidizing equivalent. Unlike HRP, a main function of catalase is the reduction of Compound I to the ferric form directly by two-electron donors such as H 2 0 2 and ethanol (159). According to the mechanism of Ishimura et al. (162) a similar reac­ tion might be expected in the case of P-450. This is an example of external mixed function oxidase (4). Now, what kind of structure would be ex­ pected for an intermediate after the oxygenated P-450 (Compound I I I type) accepts an electron from the external source? Could it be something like Compound I of catalase, HRP, or CCP? The question remains to be solved whether an intermediate species formed immediately after H R P Compound I I I accepts an electron from donor molecules is Compound I or something else. It is likely that such an intermediate possesses a trans­ ferable oxygen atom as suggested by Ullrich and Staudinger (166).

13. PEROXIDASE

4. Compound

553

III

(Oxyhemoproteins)

It is known that the oxy forms of hemoglobin, myoglobin, catalase, tryptophan pyrrolase, and H R P have similar absorption spectra. This fact would imply that the electronic structure of the oxygen bound to the heme 3 iron of these hemoproteins is essentially the same. A Fe +-0 2~ type of binding might be the case with all oxyhemoproteins. If the activation of molecular oxygen cannot be explained by the electronic structure, the proximity effect will become the probable mechanism to explain the incor­ poration of molecular oxygen into tryptophan on the surface of the trypto­ phan pyrrolase molecule (see Chapter 3 by Feigelson). Compound III of CCP has not been found. The oxygen in Compound III of H R P and milk peroxidase was found not to be dissociable in a measurable time (40, 135).

V . CONCLUSION A characteristic feature of peroxidase catalysis is known to be high specificity for the electron acceptor and extremely low specificity for the electron donor. This fact might be related to the difference in the nature of the primary product of the reaction (167). In the ordinary peroxidase reaction the free radicals of two-electron donor molecules are freed from the enzyme as a primary product, but hydroxyl radicals cannot be a primary product. Peroxidase is a kind of iron complex which can hold every chemical species present in the reduction process from O2 to H 2 0 . A rough outline of the story has now been given. The most interesting step of the 0 2 reduction on the heme iron of peroxidase seems to be the conversion from the oxy form (Compound III) to Compound I, and at present the reaction is far beyond understanding. It can be said that H R P is a useful model to analyze the mechanism of electron transfer reactions and 0 2 metabolism including oxygenase. A systematic study of the effect of heme substitution upon the various functions of H R P appears to be a promising approach. Among various donor molecules poorly specific for HRP, IAA behaves in a unique manner, like a true physiological substrate. However, the problem still remains to be solved whether the reaction arises from specific interaction between IAA molecule and the H R P protein or only from the reactivity of IAA molecule and its oxidized intermediates. Since the mechanism of IAA oxidation depends on the experimental conditions, a typical reaction of an oxygenase type would be expected under the specified conditions.

554

ISAO YAMAZAKI REFERENCES

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