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Biochem. J. (1999) 340, 579­583 (Printed in Great Britain)

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RESEARCH COMMUNICATION

Oxidation of indole-3-acetic acid by dioxygen catalysed by plant peroxidases : specificity for the enzyme structure
Pavel A. SAVITSKY*, Irina G. GAZARYAN*1, Vladimir I. TISHKOV*, L. Mark LAGRIMINI, Tautgirdas RUZGAS and Lo GORTON
*Department of Chemical Enzymology, Chemical Faculty, Moscow State University, Moscow 119899 GSP, Russia, Department of Horticulture and Crop Sciences, Ohio State University, Columbus OH 43210-1096, U.S.A., and Department of Analytical Chemistry, Chemical Centre, University of Lund, Lund, P.O. Box 124, SE 221 00, Sweden

Indole-3-acetic acid (IAA) can be oxidized via two mechanisms : a conventional hydrogen-peroxide-dependent pathway, and one that is hydrogen-peroxide-independent and requires oxygen. It has been shown here for the first time that only plant peroxidases are able to catalyse the reaction of IAA oxidation with molecular oxygen. Cytochrome c peroxidase (CcP), fungal peroxidases (manganese-dependent peroxidase, lignin peroxidase and Arthromyces ramosus peroxidase) and microperoxidase were essentially inactive towards IAA in the absence of added H O . An ## analysis of amino acid sequences allowed five structurally similar fragments to be identified in auxin-binding proteins and

plant peroxidases. The corresponding fragments in CcP and fungal peroxidases showed no similarity with auxin-binding proteins. Five structurally similar fragments form a subdomain including the catalytic centre and two residues highly conserved among ` classical ' plant peroxidases only, namely His-40 and Trp-117. The subdomain identified above with the two residues might be responsible for the oxidation of the physiological substrate of classical plant peroxidases, IAA. Key words : auxin-binding domain, auxin-binding proteins, peroxidase, structural similarity.

INTRODUCTION
Molecular mechanisms of the action of plant hormones remain a challenging problem of plant physiology and molecular biology. Plant peroxidases that are involved in stress- and pathogeninduced processes in plants [1] contribute towards molecular mechanisms of the plant immune response. Plant genomes contain numerous copies of peroxidase genes (up to 30 in Arabidopsis [2]), whose detailed functions are still unclear [3]. The changes in peroxidase expression patterns are interdependent on the level of endogenous auxins [4]. Application of exogenous auxins can induce [5] or suppress [6] peroxidase synthesis, and thus these switch either on or off peroxidase gene promoters. The whole set of physiological data indicates a key physiological role played by plant peroxidases ; however, it is still difficult to determine which substrate is the physiologically relevant one for these enzymes. Plant peroxidases (EC 1.11.1.7) catalyse the oxidation of numerous artificial and physiological electron donors (e.g. coniferyl alcohol, caffeic and ascorbic acid, etc.) utilizing hydrogen peroxide [7] : EjH O EIjH O ## # EIjS EIIjP EIIjSjH+ EjH OjP # (1) (2) (3)

acid (IAA) by a hydrogen-peroxide-independent, oxygen-requiring route catalysed by plant peroxidases [8]. The reaction mechanism is extremely complicated due to overlapping radical processes [9,10]. The recent studies on the mechanism of IAA oxidation catalysed by horseradish and tobacco (Nicotiana) peroxidases [11­13] allowed us to identify skatolyl hydroperoxide as a key reaction product, and to conclude that there should be a special binding site for IAA in plant peroxidases. We proposed that plant peroxidases are highly specific IAA oxygenases, and the reaction cycle is initiated via formation of a ternary complex, enzyme­IAA­oxygen-yielding, IAA cation radical [11] : [E­IAAkO ] (4) E$+jIAA [E­IAA]jO # # (5) [E­IAA­O ] EjIAAd+jO d- # # where IAAd+ is the IAA cation radical and O d- is the superoxide # anion radical. Further decarboxylation yields skatolyl radicals [eqn. (6)], which react with oxygen [eqn. (7)] and, subsequently, form the corresponding organic hydroperoxide [eqn. (8)]. InCH djCO (6) # # InCH djO InCH O d (7) # # ## InCH O djIAA InCH OOHjIAAd (8) ## # where IAAd is the indolyl radical, and InCH d, InCH O d and # ## InCH OOH are skatolyl radical, peroxy radical and hydroper# oxide respectively. The failure to detect compound I in the reaction course [12], and the necessity of an extra electron donor to complete the cycle [13], suggested similarities between the mechanism of action of plant peroxidases and prostaglandin H synthase [14]. The latter / has the arachidonic-acid-binding site located approx. 12 A (1.2 nm) away from the haem edge [15], i.e. we anticipated the IAAd+

where E, EI and EII are the native enzyme and its compounds I and II respectively, and S and P are respectively the substrate and the product of its one-electron oxidation. The only physiological reaction known in terms of its high specificity for the substrate is the oxidation of indole-3-acetic

Abbreviations used : CcP, cytochrome c peroxidase ; HRP, horseradish peroxidase ; IAA, indole-3-acetic acid ; LiP, lignin-dependent peroxidase from Phanerochaete chrysosporium. 1 To whom correspondence should be addressed (e-mail igazaryan!hotmail.com). # 1999 Biochemical Society


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measurements were performed over a 0.1­6 mM range of IAA concentrations in 0.1 M potassium phosphate buffer, pH 7.0, at 22 mC.

presence of a special binding site for IAA that was distinct from that involved with the binding and cleavage of hydrogen peroxide. This would permit IAA to bind on the enzyme simultaneously with indole-3-methanol, derived from skatolyl hydroperoxide [13] : EjInCH OOH EI-InCH OH EII-[InCH OH]d (9) # # # EII-[InCH OH]djIAA IAA-EII-[InCH OH]d IAAd-EII# # [InCH OH] IAAd-EIIjInCH OH (10) # # The goal of the present study was to test and to examine all available haem-containing peroxidases in the reaction of IAA oxidation with molecular dioxygen to examine whether there is any similarity between haem-containing peroxidases and auxinbinding proteins. This approach has allowed us to show for the first time both the reaction specificity of the enzyme structure and the presence of structurally similar fragments in classical plant peroxidases and auxin-binding proteins.

Homology studies
Amino acid sequences of seven auxin-binding proteins were taken from GenBank (accession nos. are given in parentheses) : Arabidopsis thaliana (X69901), Capsicum annum (Z48451), Malus domestica (U77952), maize (L08425, L08426) and Nicotiana tabacum (X70902, X70903). Alignments of amino acid sequences of haem-containing peroxidases were adapted from reviews published previously [17,18]. The sequence of anionic soya-bean peroxidase was taken from GenBank (AF007211). The search for structurally similar fragments was performed by using the MASAW program, version 2.0.0, with the BLAST algorithm [19]. Mapping of structurally similar fragments was performed by using the crystal structure of HRP [20] and RASMOL, version 2.6.

MATERIALS AND METHODS Chemicals
IAA, iron(II) chloride, microperoxidase MP-11, and haemin were purchased from Sigma, St. Louis, MO, U.S.A., indole-3methanol and indole-3-aldehyde were from Aldrich, Milwaukee, WI, U.S.A., and hydrogen peroxide and its salts were from Merck, Darmstadt, Germany. Milli Q water was used for the preparation of all solutions.

RESULTS AND DISCUSSION IAA oxidation by oxygen catalysed by haem-containing peroxidases
IAA is known to be degraded in water solutions. However, in the absence of catalysts the process was very slow. In deionized water, IAA was relatively stable at 4 mC, and no degradation products were detected by HPLC for at least 24 h. Traces of IAA degradation products can be recorded after 5­6 h of incubation in distilled water at room temperature. To degrade IAA without the addition of plant peroxidases, one may use iron(II) chloride or haemin in a highly acidic medium [8]. In 1 % (v\v) acetic acid and in the presence of haemin (0.01 mg\ml), 1 mM IAA was degraded by 50 % in 1 h, yielding non-identified polymeric products. Iron(II) chloride (0.05 mg\ml) was also an efficient catalyst of IAA degradation under the above conditions. Methylene-3-oxindole is a major degradation end product when iron(II) chloride is used as a catalyst. The product spectrum produced during the course of degradation closely resembled that described for both horseradish and tobacco peroxidases [13], showing the presence of indole-3-methanol, indole-3-aldehyde, oxindole-3carbinol and some minor non-identified products (results not shown).

Enzymes
Horseradish peroxidase (HRP) isoenzyme C was purchased from Biozyme Laboratories Ltd., San Diego, CA, U.S.A, and used without further purification. Soya-bean peroxidase, RZ 1.1 (where RZ denotes the ratio of the Soret band absorbance to A ), was purchased from Enzymology Inc., Columbus, OH, #)! U.S.A., and was purified further by anion-exchange FPLC. Arthromyces ramosus (Coprinus cinereus) peroxidase was purchased from Suntory Ltd., Kyoto, Japan. Anionic tobacco peroxidase was purified from leaves of Nicotiana syl estris plants, which overexpress this enzyme [16]. Lignin peroxidase H2 (LiP) and Mn-peroxidase H5 from Phanerochaete chrysosporium were kindly provided by Professor Ming Tien (Pennsylvania State University, State College, PA, U.S.A.), and cationic peanut peroxidase was generously given by Professor R. B. van Huystee (University of Western Ontario, London, Canada). Wild-type recombinant yeast cytochrome c peroxidase (CcP) expressed in Escherichia coli was obtained from Professor A. G. Cass (Imperial College, London, U.K.).

Table 1 Oxygen consumption in the presence of 1 mM IAA in 0.1 M potassium phosphate buffer, pH 7.0, at 22 mC in the presence of 30 µMH2O2
The activity in the absence of hydrogen peroxide is shown in parentheses. ARP, fungal peroxidase from Arth. ramosus ; MnP, manganese-dependent peroxidase from Ph. chrysosporium ; PNP, cationic peanut peroxidase ; SBP, soya-bean peroxidase ; TOP, anionic tobacco peroxidase. Concentration added (µM) 2.0 1.0 1.0 1.5 30 0.8 0.7 2.0 1.5 Initial rate (v ; µM/min) 15 (0) 25 (0) 15 (0) 30 (0) 1.5 (0) 10 (10) 30 (14) 48 (2.0) 2.5 (2.5) Duration of reaction (min) 2 2 2 1 20 20 10 40 80

HPLC studies
Incubations were performed in 0.1 M potassium phosphate buffer, pH 7.0, at 22 mC unless stated otherwise. The reaction was initiated by the addition of enzyme ; 200-µl aliquots were then taken and analysed by reverse-phase HPLC on a C Columbus ") / column (300 A, 5 µM, 150 mmi4.6 mm ; Phenomenex, Torrance, CA, U.S.A.) by using isocratic elution in a mixture of methanol\1 % acetic acid (2 : 3, v\v) at a flow rate of 1.0 ml\min. A Shimadzu LC-5A instrument was used in the experiments for measurement of A . #&!

Enzyme CcP LiP MnP ARP Microperoxidase HRP TOP SBP PNP

O2 consumed ( %) 15 25 15 15 15 100 100 100 100 (0) (0) (0) (0) (0) (100) (100) (100) (100)

Oxygen uptake
Studies were performed with an oxygen electrode fitted with a plastic chamber (Rank Brothers, Cambridge, U.K.). Kinetic
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(20) (13) (100) (80)


Structural similarity between peroxidases and auxin-binding proteins

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Figure 1

Amino acid sequences similar for both auxin-binding proteins and plant peroxidases

Auxin-binding proteins studied were those from A. thaliana, Cap. annum, M. domestica, maize and N. tabacum. ARP, fungal peroxidase from Arth. ramosus ; HRPC, horseradish peroxidase isoenzyme C ; MnP, manganese-dependent peroxidase from P. chrysosporium ; PNP, cationic peanut peroxidase ; SBP, soya-bean peroxidase ; TOP, anionic tobacco peroxidase.

Fungal and yeast peroxidases were almost inactive at pH 3.5 compared with the horseradish enzyme, e.g. complete degradation of 100 µM IAA with 30 nM HRP was achieved in 30 min, whereas LiP and CcP, under similar conditions, showed only 2 % conversion. The principal difference between the spectra of degradation products for the oxygenase reaction catalysed by plant and fungal peroxidases was the presence of indole-3methanol for the former. This indicated the inability of fungal peroxidases to catalyse reactions consuming skatolyl hydroperoxide, which are shown in eqns. (9) and (10). To avoid non-specific radical reactions of IAA degradation occurring in acid medium, we performed the experiment under neutral conditions, i.e. at pH 7.0. Since the rate of oxygen uptake was shown to correspond to that of IAA degradation [21], we followed the kinetics of oxygen consumption. In neutral media, both CcP and fungal peroxidases were essentially inactive towards IAA, as determined by HPLC and oxygen-uptake measurements (Table 1). The addition of hydrogen peroxide should activate the common peroxidase pathway represented by eqns. (1)­(3), and IAA cation radicals formed should eventually yield skatolyl hydro-

peroxide via reactions (6)­(8). This could help to activate reactions (9) and (10), and thereby stimulate oxygen consumption, as has been shown previously for tobacco peroxidase [21]. As assessed by measurements of oxygen uptake in the presence of 1 mM IAA and in both the absence and presence of added hydrogen peroxide [catalysed by different haem-containing peroxidases (Table 1)], only plant peroxidases were able to catalyse the reaction without added hydrogen peroxide. However, in the case of anionic plant peroxidases (tobacco and soya bean), hydrogen peroxide served to remove an initial ` lag ' period, indicating the differences in the initiation mechanism between anionic and cationic peroxidases. Oxygen consumption in the case of CcP and fungal peroxidases was observed only in the presence of hydrogen peroxide. Its duration was rather short, and the amount of oxygen consumed was approx. 1­2 mol. equivalents with respect to the amount of hydrogen peroxide added to the mixture. Thus the oxygen consumption in this case was due to the formation of IAA radicals via reactions (1)­(3), and their subsequent interaction with molecular oxygen via reaction (7). Skatolyl hydroperoxide, formed as shown in reaction (8), did not activate the oxygenase cycle. This suggests either an
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Figure 2

Fragments structurally similar to those found in auxin-binding proteins for HRP

Fragments structurally similar to those found in auxin-binding proteins are shown in the structure of HRP coloured red (amino acids 39­44), light blue (amino acids 114­133), green (amino acids 96­114), yellow (amino acids 71­82) and magenta (amino acids 4­18). Trp-117 is shown in dark blue and His-40 is in red. The peroxidase molecule is shown from the distal site.

inability of skatolyl hydroperoxide to act as an oxidative substrate, or an absence of a special binding site for IAA that would permit the reaction sequence (9) and (10). The data show unequivocally the absence of a hydrogen-peroxide-independent mode of activity towards IAA for microperoxidase and CcP and fungal peroxidases.

Structural similarity between auxin-binding proteins and classical plant peroxidases
The specificity of IAA oxidation for the enzyme structure, as described in the present study, enticed us to look for the structural basis for this phenomenon. Fortunately, both amino acid sequences and crystal structures are available for CcP [22], lignin [23] and Mn-peroxidases [24], Cop. cinereus [25] (Arth. ramosus [26]) peroxidase, peanut peroxidase [27] and HRP [20]. The peculiarities of the reaction mechanism, and the inability of prokaryotic and fungal peroxidases to catalyse the reaction of IAA oxidation, may be easily rationalized if there is a specific IAA-binding site in classical plant peroxidases. The search for structural similarity between haem-containing peroxidases and auxin-binding proteins performed for the first time in the present study allowed us to identify five structurally similar fragments (Figure 1). It is evident that only classical plant peroxidases, i.e. tobacco, horseradish, soya-bean and peanut peroxidases, have fragments structurally similar to those of auxin-binding proteins. The corresponding fragments in CcP and fungal peroxidases show no similarity to auxin-binding proteins. Unfortunately, there is no information on the structures
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of the sequenced auxin-binding proteins, and thus the following discussion is on the basis of the structure of plant peroxidases. Five structurally similar fragments presented in Figure 1 (I­V) form a subdomain (helices A, B, D­Dh and C) in the distal domain of plant peroxidase (Figure 2). The first structurally similar fragment (fragment I in Figure 1, and shown in red in Figure 2) corresponds to the sequence co-ordinating the haem from the distal site of the haem-binding pocket. This sequence belongs to helix B, and is highly conserved among all haemcontaining peroxidases. However, only classical plant peroxidases contain two histidine residues in this fragment, namely His-40 and His-42. The sequences flanking this fragment in auxin-binding proteins are similar to the loop between helices D and Dh in plant peroxidases (residues 114­133 of fragment II in Figure 1, and shown in blue in Figure 2). This loop contains the tryptophan residue highly conserved among all classical plant peroxidases [17]. In the HRP crystal structure, Trp-117 is approx. / 8­9 A (0.8­0.9 nm) apart from nitrogen atoms of the porphyrin ring, and is in the zone of electron tunnelling. The fluorescence of the protein tryptophan residue has been recorded during the course of tyrosine peroxidation catalysed by HRP [28], and it has been suggested that it plays an important role in electron transfer through the peroxidase molecule. Auxin-binding proteins also contain the fragment similar to helix D (residues 97­114 of fragment II in Figure 1 ; shown in green in Figure 2). Two other structurally similar fragments correspond to helix A (residues 4­18 of fragment IV, Figure 1 ; shown in magenta in Figure 2) and the beginning of helix C (residues 71­82 of fragment V in Figure 1 ; shown in yellow in


Structural similarity between peroxidases and auxin-binding proteins
Figure 2) in the structure of HRP. Taken together, these five fragments form a subdomain including the catalytic centre and two residues highly conserved among classical plant peroxidases only, namely His-40 and Trp-117. The identified domain containing the above two residues could be directly involved in the oxidation of the physiological substrate of classical plant peroxidases, IAA. This work in part was supported by a grant from the European project (BIO4-CT972199) and The Swedish Royal Academy of Science (grant 12552).
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Received 16 March 1999/19 April 1999 ; accepted 27 April 1999

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