Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Abstract

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to catalyze the oxidation of indole by H 2 O 2 , with generation of 2-and 3-oxoindole as the major products. This reaction occurred in the absence of O 2 and reducing agents and was not inhibited by superoxide dismutase or hydroxyl radical scavengers, although it was strongly inhibited by L-Trp. The stoichiometry of the reaction indicated a one-to-one correspondence for the consumption of indole and H 2 O 2 . The 18 O-labeling experiments indicated that the oxygen incorporated into the monooxygenated products was derived almost exclusively from H 2 18 O 2 , suggesting that electron transfer was coupled to the transfer of oxygen from a ferryl intermediate of IDO. These results demonstrate that IDO oxidizes indole by means of a previously unrecognized peroxygenase activity. We conclude that IDO inserts oxygen into indole in a reaction that is mechanistically analogous to the "peroxide shunt" pathway of cytochrome P450.monooxygenase | oxygen isotopic labeling T he heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-determining step of L-tryptophan metabolism in nonhepatic mammalian tissues by inserting both atoms of O 2 into the indole ring to form N-formylkynurenine (N-FK) (1). This dioxygenase activity of IDO requires O 2 and the reduced (Fe 2+ ) enzyme or O 2•− and the oxidized (Fe 3+ ) enzyme. In recent years, an increasing number of physiological consequences of IDO activity have been identified, including links to neural, ocular, and immunological disorders (2). Of these roles, the ability of IDO expressed by tumors to suppress the normal response of T lymphocytes and enable tumor survival and growth has attracted the most intense interest, owing to its implications for the development of new therapeutic approaches in cancer treatment (3).Aside from tryptophan (Trp), the dioxygenase activity of IDO extends to oxidation of other indoleamines, such as 5-hydroxy-LTrp, serotonin, and tryptamine, whereas indole, 3-methylindole, and indoleacetic acid are not substrates (4-6). Although indole can apparently bind to the oxygenated enzyme (IDOFe 3+ -O 2•− ) or to the IDOFe 2+ -CO complex (7), autoxidation to IDOFe 3+ is unaffected by the presence of indole, and oxidation of indole does not occur (5). Notably, IDO does not catalyze oxidation of L-Trp by H 2 O 2 (4, 8, 9). Consequently, the reactivity of IDO with H 2 O 2 received little attention for more than 30 years. During that time, IDO was noted to have peroxidase activity (6) and to catalyze the H 2 O 2 -supported N-demethylation of benzphetamine and hydroxylation of aniline (10), but these activities were not studied further.Recently, reactivity of the enzyme with H 2 O 2 has received considerable attention in light of spectroscopic detection of a compound II-like ferryl species (11, 12) and quantum mechanics/ molecular mechanics simulations implicating a role for this intermediate in the catalytic cycle (13). Subsequently, the peroxidase activity of IDO has received renewed atten...

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions

“…The observation that oxindolylalanine formation is O 2 -independent is consistent with a role for IDO's peroxygenase activity (70), in which the two-electron reduction of compound I is tightly coupled to the transfer of the ferryl oxygen into a substrate, in this case, the insertion of the oxygen atom into the indole ring of L-Trp to form oxindolylalanine (Scheme 2, reaction C). As indicated above, the efficiency of this putative peroxygenase activity with L-Trp as a substrate is markedly less when compared with indole as a substrate (70). Our spectroscopic data indicate that in the presence of H 2 O 2 and L-Trp, the majority of IDO compound I undergoes a one-electron reduction to form compound II.…”

“…Lu and Yeh (26) frequently exposed rIDO to high levels of H 2 O 2 (Ͼ600:1 mol/mol) and L-Trp (Ͼ1000:1 mol/mol), conditions that may favor compound II formation with which L-Trp does not react. This apparent discrepancy may also reflect that whereas L-Trp is oxidized by IDO in the presence of H 2 O 2 , it is only to a minor extent, especially compared with indole as a substrate for IDO in the presence of H 2 O 2 (70). Thus, Kuo and Mauk (70) recently reported that 0.5 M rIDO in the presence of excess indole and H 2 O 2 oxidized ϳ222 M indole within 5 min of reaction.…”

“…The oxidation of L-Trp is complex with different one-and two-electron oxidants yielding an array of oxidation products, including the tryptophanyl radical, hydroxytryptophans, oxindolylalanine, dioxindolylalanine, N-formylkynurenine, kynurenine, and hydroxylated kynurenines, with these products commonly accounting for a portion of the total amount of L-Trp oxidized (71)(72)(73)(74)(75) (71), whereas the ability of IDO peroxygenase to oxidize indole into oxindoles and analogues of N-formylkynurenine is O 2 -independent (70).…”

“…Although the oxidation of indole by IDO peroxygenase yields oxindoles as the major product, analogues of N-formylkynurenine are also formed (70). As such, it is possible that the O 2 -insensitive portion of kynurenine formed by IDO in the presence of H 2 O 2 and L-Trp is derived, in part, from the putative peroxygenase activity.…”

Enzymatic aerobic ring rearrangement of optically active furylcarbinols

“…2a), implying at least the participation of a protein-bound oxidizing species. The strongest indication for the non-mediated furan oxidation through compound I, or alternatively through a hydroperoxoferric intermediate 31 , arose from experiments using isotopically labelled oxygen as terminal oxidant 32 . Ring expansion by the GOx/CPO system in an argon atmosphere enriched in heavy oxygen (20 vol% 18 O 2 ) gave rise to the mono- 18 O-marked product rac-2a- 18 O in high isotopic yield (Fig.…”