The Roles of Superoxide Anion and Methylene Blue in the Reductive Activation of Indoleamine 2,3-Dioxygenase by Ascorbic Acid or by Xanthine Oxidase-Hypoxanthine*

To clarify the roles of superoxide anion (0;) and methylene blue in the reductive activation of the heme protein indoleamine 2,3-dioxygenase, effects of xan- thine oxidase-hypoxanthine used at various oxidase concentration levels as an 0; source and an electron donor on the catalytic activity of the dioxygenase have been examined in the presence and absence of either methylene blue or superoxide dismutase using L- and D-tryptophan as substrates. In the absence of methyl- ene blue, initial rates of the product N-formylkynuren-ine formation are enhanced in parallel with the xan- thine oxidase level up to -100 and -50% of the apparent maximal activity (-2 s-’) for L- and D-Trp, re- spectively. Superoxide dismutase effectively inhibits the reactions by 80-98% for both isomers. Additions of methylene blue (25 p ~ ) help to maintain the linearity of the product formation that would be rapidly lost a few minutes after the start of the reaction without the dye, especially for L-Trp. Additions of methylene blue also enhance the activity to the maximal level for D-Trp. In the presence of methylene blue, the inhibitory effects of superoxide dismutase are considerably de-creased with the increase in xanthine oxidase concen- tration, and at near maximal dioxygenase activity levels superoxide dismutase is totally without effect. In separate anaerobic experiments leuco-methylene blue,


The Roles of Superoxide Anion and Methylene Blue in the Reductive Activation of Indoleamine 2,3-Dioxygenase by Ascorbic Acid or by
Xanthine Oxidase-Hypoxanthine* (Received for publication, July 6, 1988)

Masanori Sono
From the Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208 To clarify the roles of superoxide anion (0;) and methylene blue in the reductive activation of the heme protein indoleamine 2,3-dioxygenase, effects of xanthine oxidase-hypoxanthine used at various oxidase concentration levels as an 0; source and an electron donor on the catalytic activity of the dioxygenase have been examined in the presence and absence of either methylene blue or superoxide dismutase using L-and D-tryptophan as substrates. In the absence of methylene blue, initial rates of the product N-formylkynurenine formation are enhanced in parallel with the xanthine oxidase level up to -100 and -50% of the apparent maximal activity (-2 s-') for L-and D-Trp, respectively. Superoxide dismutase effectively inhibits the reactions by 80-98% for both isomers. Additions of methylene blue (25 p~) help to maintain the linearity of the product formation that would be rapidly lost a few minutes after the start of the reaction without the dye, especially for L-Trp. Additions of methylene blue also enhance the activity to the maximal level for D-Trp. In the presence of methylene blue, the inhibitory effects of superoxide dismutase are considerably decreased with the increase in xanthine oxidase concentration, and at near maximal dioxygenase activity levels superoxide dismutase is totally without effect. In separate anaerobic experiments leuco-methylene blue, generated either by photoreduction or by ascorbate reduction, is shown to be able to reduce the ferric dioxygenase up to 25-40%. Substrate Trp and heme ligands (CO, n-butyl isocyanide) help to shift a ferric form t , ferrous form equilibrium to the right. Thus, under aerobic conditions leuco-methylene blue might similarly be able to reduce the dioxygenase in the presence of an electron donor with the aid of substrate and 02. These results strongly suggest that indoleamine 2,3-dioxygenase can be activated through different pathways either by 0; or by an electron donor-methylene blue system. For the latter case, the dye is acting as an electron mediator from the donor to the ferric dioxygenase.
The apparent involvement of superoxide anion (0;) in the activation and catalytic processes of the heme protein, indole-amine 2,3-dioxygenase, has been an intriguing property of this dioxygenase (1-3) since its discovery in Hayaishi's laboratory (4, 5). This unique feature of the enzyme along with its molecular (monomeric glycoprotein, M , -41,000) (6) and immunogenic (7) properties, relatively wide substrate specificity for several indoleamines (6,8), and ubiquitous mammalian tissue distribution except the liver (3, 9-11), clearly distinguish indoleamine 2,3-dioxygenase from another similar heme-containing protein, tryptophan 2,3-dioxygenase (12).l The catalytic reaction of indoleamine 2,3-dioxygenase can be initiated by 0; that is added directly using KO2 (14) or generated either chemically (1, 2) or enzymatically (2). Superoxide dismutase was reported to significantly inhibit the reaction not only at the initial stage but also at the steady state (1). The observed dioxygenase activation by 0; is directly related to the fact that the enzyme is in the ferric (inactive) state when purified from tissues ( 5 ) and needs to be reduced to the ferrous (active) state to perform the catalytic reaction (15). Unlike several other 02-binding heme proteins such as hemoglobin (16, 17), myoglobin (16), and cytochrome P-450,,,,z the ferric native form of indoleamine 2,3-dioxygenase readily binds 0, to form the dioxygen complex of the ferrous enzyme, which is also catalytically active but highly autoxidizable in the presence of substrate (18). However, superoxide anion alone is not sufficient to maintain the fully activated state of the dioxygenase, and another co-factor, an artificial dye, methylene blue, is also required for maximal activity (14). Methylene blue has been used in the following two typical assay systems for the dioxygenase: ( a ) ascorbatemethylene blue ( 5 ) and (6) KOz-methylene blue (14). In system a, the more convenient assay system, the dye is absolutely required for the catalytic reaction since ascorbate alone can hardly activate the enzyme. Methylene blue here is considered to be an electron carrier from ascorbate to molecular oxygen to generate 0, (1, 2). In system b, methylene blue is required to maintain the initial linearity of the reaction that is quickly lost in 1-2 min after the start of the reaction (14). The roles of methylene blue in these two systems thus seem to differ. Yet, no clear explanations for the actual role(s) of the dye in relation to the role of 0; have been offered.
To address this question in the present study an alternative 0;-generating enzymatic system, xanthine oxidase-hypoxanthine, has been used for the activation of indoleamine 2,3dioxygenase. The advantages of using this system are that the rate of 0; generation can be easily controlled by changing the concentration of the oxidase and that the catalytic reaction can be continuously monitored by following absorbance ' Copper was later shown to be nonessential for the catalytic Examined by a pulse radiolysis technique by K. Kobayashi (Osaka activity of tryptophan 2,3-dioxygenase (13).
University, Osaka, Japan), personal communication. changes due to the product formation. Xanthine oxidase can also be used as an electron donor. Since methylene blue is a good electron acceptor from xanthine oxidase (19,20), as is molecular oxygen, it is necessary to carry out experiments both in the presence and absence of the dye in order to examine separately the effects of 0; and the actual role of methylene blue in the xanthine oxidase-methylene blue cofactor system. In view of these points, effects of a relatively wide range (1.7-40 milliunits/ml) of xanthine oxidase levels on catalytic activity of indoleamine 2,3-dioxygenase has been examined in detail in this study in the presence and absence of either methylene blue or superoxide dismutase using L-TV and ~-T r p as dioxygenase substrates. This study has allowed quantitative analysis of the correlation between the extent of the 0; participation and the reductive activation of indoleamine 2,3-dioxygenase and has provided new evidence that methylene blue acts as an electron mediator from donors to the ferric dioxygenase.

EXPERIMENTAL PROCEDURES
Enzymes-Indoleamine 2,3-dioxygenase was purified from rabbit small intestine by the method of Shimizu et al. (6), except that the final isoelectrofocusing was omitted and instead step 6 (Sephadex G-100 chromatography) was repeated 2-4 times. The purified native ferric enzyme exhibited an A406/A280 value of 1.7-1.8 in 20 mM potassium phosphate buffer a t p H 6.0 and 24 "C and was 60-70% pure as judged by sodium dodecyl sulfate gel electrophoresis (21). The amount of the enzyme was expressed in terms of its heme content based on the absorbance at 406 nm (c = 159 mM" cm" a t p H 6.0 and 25 "C (21)). Xanthine oxidase from cow milk and catalase from beef liver were products of Boehringer Mannheim. Xanthine oxidase activity was determined by the method of Kalcker with hypoxanthine as substrate (22,23). One unit is defined as the amount of enzyme which catalyzes the formation of 1 pmol of uric acid/min in a total volume of 1.0 ml at 24 "C and pH 7.7 using an 6290 value of 12.2 mM" cm" (23). Superoxide dismut.ase purified from bovine erythrocytes with a specific activity of 3300 units/mg of protein (15, 24) was a generous gift of Professor Osamu Hayaishi (Kyoto University).
Materials-Sperm whale myoglobin (Type 11) was purchased from Sigma. All of the following chemicals of reagent grade obtained from the companies indicated were used without further purification: ascorbic acid and hypoxanthine from Aldrich; L-and ~-T r p from Sigma; methylene blue, isopropanol, and acetophenone from Fisher; methyl viologen from ICN Pharmaceuticals.
Photoreduction Method-Anaerobic reduction of artificial electron carriers (methylene blue and methyl viologen) or heme proteins (indoleamine 2,3-dioxygenase and myoglobin) was performed by photoreduction under argon according to the method reported by Ward and Chang (25). Trace amounts of dioxygen in the argon were removed by bubbling argon through an acidic aqueous Cr(C104)2 solution containing amalgamated zinc (26). Samples to be photoreduced were prepared as follows: buffer (0.1 M potassium phosphate, pH 7.0) containing 2% (v/v) isopropanol and -0.1% (v/v) acetophenone was placed in a rubber septum-stoppered cuvette (path length 0.2 cm) and made anaerobic by gently bubbling with argon gas on ice for a t least 30 min through a syringe needle inserted through the rubber septum. Next, a microliter volume of a sample from its concentrated aqueous stock solution (0.5-1 mM) was placed in a small side reservoir attached near the top of the cuvette, and the bubbling was further continued for another 10-15 min. Then, the sample was mixed in the buffer by tilting the cuvette. The anaerobic solution of the sample thus prepared was subjected to photoreduction in a circulating icewater bath to protect the sample from heat while keeping slight positive pressure with argon in the cuvette. The photoreduction apparatus consisted of a 400-watt quartz mercury arc immersion UV lamp (Ace Glass Inc.) and a power supply. The reduction of the sample was monitored from its spectral changes after each photoirradiation.
Spectrophotometric Measurements-The measurements were carried out with either a Union Giken SM-401 spectrophotometer or a Varian Cary 219 spectrophotometer each of which was equipped with a circulator for temperature control (25 f 1 "C). Except for the photoreduction experiments (see above), all measurements were done in a cuvette with a 1-cm light path. Fig. 1 shows typical time courses for the formation of the product, N-formylkynurenine (X, , , = 321 nm) using the lower two xanthine oxidase concentration levels (1.7 and 3.4 milliunits/ml) examined in this study. Throughout the text, concentrations of xanthine oxidase and of superoxide dismutase are expressed as their catalytic activity values rather than ordinary molar concentration values. Catalase is also added to prevent the dioxygenase from decomposition by the hydrogen peroxide that is produced in the system as a byproduct (18). Even though both ~-T r p (0.2 mM) and ~-T r p (5 mM) are used in sufficient concentrations (>50 K,) (6, 27), one notices that the L-isomer yields considerably higher initial rates (V,) for the product formation than the D-isomer by a factor of about 1.7. This trend is consistently observed for higher oxidase concentrations. It should be pointed out that the Vo values for L-TW and ~-T r p under these conditions account for only 6-10 and 4-6%, respectively, of the apparent maximal turnover number (2 s-l) (6, 15). Interestingly, however, the linearity of the time course for L -T~ is gradually lost after 3-4 min following the start of the reaction, while relatively good linearity is held for 25 min for the D-isomer. Results similar to those for ~-T r p were observed with D L -T~~ using KOn as an 0; source (14), but ~-T r p was not previously examined separately from the L-isomer in the absence of methylene blue. When methylene blue (25 PM) is present, different effects are seen for the two Trp isomers. The dye helps to maintain the initial linearity for ~-T r p without significantly changing the initial rate of the product formation (not shown), while for ~-T r p , methylene blue not only improves the linearity but also markedly enhances the catalytic rate by a factor of 2-4 (see below).

RESULTS
In Fig. 2, the inhibitory effects of varying concentrations (1-880 units/ml) of superoxide dismutase on the Vo values for L-and ~-T r p using the lowest xanthine oxidase concentration level (1.7 milliunits/ml) examined are shown in the absence ( A ) and presence ( B ) of methylene blue. In the absence of the dye, superoxide dismutase effectively inhibits the product formation; 1 unit of dismutase causes 55-65% inhibition, and nearly complete (-98%) inhibition is achieved with 350 units of the enzyme. Both L-and D -T~ exhibit similar results. Thus, 0; is the sole and key activator under these conditions. However, when methylene blue (25 PM) is added under the same conditions, the maximal inhibition by superoxide dismutase is noticeably reduced to 45-48%, and 1 unit of dismutase causes only -7% (~-T r p ) a n d -17% (L-  (0) in the presence and absence of methylene blue. Boiled superoxide dismutase (100 "C, 5 min) was also examined for A (1 unit/ml) and B (20 units/ml).

Trp) inhibition. This indicates that about
one-half of the activation is attributed to OH and the remaining half to an OH-independent reduction pathway(s). Boiled superoxide dismutase exhibits no inhibition with or without methylene blue.
Since such effects of methylene blue as observed in this study in a xanthine oxidase and its substrate system have not previously been reported, extents of superoxide dismutase inhibition in the presence of the dye is further examined as a function of xanthine oxidase concentration. Results with L-Trp are demonstrated in Fig. 3. Although the rate of the product formation (open circles) increases with the increase in xanthine oxidase concentration, the extent of inhibition by dismutase becomes significantly smaller at higher oxidase concentrations. At 10 milliunits/ml xanthine oxidase, inhibitory effects of superoxide dismutase are negligible. Similar results are obtained with D-TV except that a somewhat higher xanthine oxidase concentration (30 milliunits/ml) is required to completely diminish the inhibition by superoxide dismutase (see Fig. 4B). These values are in good agreement with the previously reported apparent maximal turnover number of -2 s-l (6, 15). The slightly lower value for L-TV than for ~-T r p is probably due to either the L-isomer-specific substrate inhibition ( 5 ) or to the non-optimal pH value for ~-T r p (6). that in the presence of the dye, the slopes of the linear time courses for the steady state product formation rates ( V) rather than Vo values are plotted here; the V values are slightly smaller than the Vo values.
It is also noted that even in the absence of methylene blue, superoxide dismutase-insensitive activity (closed squares) is almost linearly enhanced for both L-and D-TW cases when xanthine oxidase concentration is raised. When 40 milliunits/ ml oxidase is used, -20% of the maximal activity remains uninhibited by superoxide dismutase for both substrates, indicating that direct reduction of the dioxygenase by xanthine oxidase is occurring to some extent under these conditions. The addition of methylene blue (25 PM) completely diminishes the inhibitory effects of superoxide dismutase (solid circles) at xanthine oxidase concentrations of 10 milliunits/ml for L-Trp and 30 milliunits/ml for D -T I~, where -40 and -90% of the apparent maximal activity are attained. These results strongly suggest that either in the presence or absence of methylene blue, some portions of the dioxygenase activation are not mediated by 0,. The activation process is entirely independent of O,, especially when the dioxygenase is turning over at its near-maximal rate (-2 s-l) for both substrates in the presence of methylene blue. The most likely reductant of the dioxygenase in such cases is the reduced methylene blue, i.e. leuco-methylene blue; the dye is known to be a good electron acceptor from xanthine oxidase under both anaerobic and aerobic conditions (19,20).
To test this possibility, the following two series of experiments have been carried out where leuco-methylene blue is mixed with the ferric dioxygenase under anaerobic conditions. In the first series, methylene blue is reduced by a photoreduction method as described under "Experimental Procedures." Spectral changes during the course of the experiments are shown in Fig. 5. Photoirradiation for 7-10 min is sufficient to completely convert the dye (blue color) to its reduced form (colorless) (spectrum a "-* b). When ferric indoleamine 2,3dioxygenase is mixed in the leuco-methylene blue solution thus prepared under argon, a spectral change (Fe" * c ) occurs that is indicative of the partial reduction of the ferric enzyme as judged from an isosbestic point (-416 nm) for the ferric and ferrous enzyme. The extent of the reduction under these conditions reaches only about 25%. An addition of L-Trp (0.25 mM) somewhat but significantly raises the extent of the reduction from -25 to -40% (c versus d ) . In both the presence and absence of the substrate, bubbling CO through the samples almost completely converts the enzyme to its ferrous-CO form (not shown); an addition of dithionite to FIG. 5. Spectral changes upon reduction of ferric indoleamine 2,3-dioxygenase with photoreduced methylene blue under anaerobic conditions. An anaerobic 25 PM methylene blue solution (spectrum a, dotted line) was prepared first in a total volume of 300 pl of 0.1 M potassium phosphate buffer, p H 7.0, at 25 "C in a 0.2-cm cuvette as described under "Experimental Procedures." This solution was photoirradiated a t 2 5 "C for 7 min to completely reduce methylene blue to leuco-methylene blue (spectrum b, short dashed line). Then, several microliters of a concentrated (-450 j" stock solution of indoleamine 2,3-dioxygenase (final 5.5 PM) were anaerobically added to the leuco-methylene blue solution at 25 "C. The spectrum c (dashed-dotted line) was recorded when the spectral changes reached equilibrium at about 45 min after mixing the solution. Spectrum d (solid line) corrected for 5.5 FM heme concentration was obtained from separate experiments in which 0.25 mM ~-T r p was added to the reaction mixture after the ferric dioxygenase had been mixed with the leuco-methylene blue solution. Spectra of the native ferric dioxygenase (Fe", dashed-double-dotted line) and its reduced form (Fe", dashed line) overplotted in the Soret region after correction to 5.5 PM heme concentration were also obtained from separate experiments. The former was converted to the latter by photoreduction in 2 min. relatively high absorbance values, especially notable in the 620-720nm region as compared with those in Fig. 5, are due to the formation of undissolved particles of leuco-methylene blue in the sample. This was caused by the conversion of the relatively high concentration (125 PM) of methylene blue that was soluble (monocationic form) in aqueous solution, to leuco-methylene blue that was less soluble (neutral form) under these conditions. ensure the reduction of the enzyme causes only a small further spectral change. The resulting ferrous-CO enzyme exhibits a Soret peak at 420 nm (-Trp) or 418 nm (+0.5 mM ~-T r p ) . Similar spectral conversion to near complete ferrous enzymeligand complex is also observed with n-butyl isocyanide, a heme ligand which binds tightly to the ferrous enzyme ( K d S lop6 M).3 For comparison, when sperm whale myoglobin (10 p~) is used in place of the dioxygenase in the presence of 25 p~ leuco-methylene blue, over 95% reduction is achieved without adding any heme ligand under the same experimental conditions (results not shown). In separate experiments, when methylene blue is replaced by methyl viologen (25 p~) , the reduction of the dioxygenase is complete within a few minutes.
In the second series of experiments, methylene blue is chemically reduced by ascorbic acid (10 mM) under anaerobic conditions. Spectral changes during the experiments are shown in Fig. 6. Since the reduction of the ferric dioxygenase in the presence of L-TT with 25 p~ leuco-methylene blue does not exceed 40% in the experiments described above, the concentration of the dye is raised to 125 pM in this case. Anaerobic reduction of methylene blue with ascorbate is relatively slow under the conditions employed, but nearly complete (-98.5%) reduction can be achieved in 1 h (spectrum a). Mixing of ferric dioxygenase with ascorbate-leuco-methylene blue solution causes spectral changes similar to those observed above (Fig. 5). The extents of the reduction in this case, however, are considerably greater either with (-65%, spect r u m e ) or without (-40%, spectrum b ) added L-TW (0.33 mM). The reduction (c "-* d + e ) is very slow. The greater reduction is probably due to the higher concentration of leucomethylene blue in the media. Bubbling with CO results in the nearly complete formation of the ferrous-CO enzyme ( A, , , = M. Sono, unpublished results. 418 nm, spectrum not shown); only a small further spectral change is seen upon addition of dithionite.

DISCUSSION
A significant finding in this study is that, under certain conditions, 0; is not an absolute requirement for the maximal catalytic activity of indoleamine 2,3-dioxygenase. This conclusion is supported by the results shown in Figs. 3 and 4 which indicate that in the presence of methylene blue (25 PM) the near-maximal activity that is attained using xanthine oxidase is hardly inhibited by superoxide dismutase added in sufficiently high concentrations (880 units/ml). It is unlikely that methylene blue directly prevents the superoxide dismutase activity since the dismutase can still inhibit the dioxygenase catalytic reaction when used with low levels of xanthine oxidase even in the presence of the dye at the same concentration (25 p M ) (Fig. 2B). In the ascorbate-methylene blue co-factor system, inhibitory effects of superoxide dismutase (maximum about 50%) were reported to be significantly diminished in parallel with the increases in the dye concentration and in parallel with the increase in the catalytic activity of the dioxygenase (2). When 25 p~ methylene blue is used in the presence of 10 mM ascorbate, no inhibition is detected in the present study even with 880 units/ml superoxide dismutase (not shown). Obviously, methylene blue is by-passing the 0;-mediated dioxygenase activation pathway in both ascorbate-methylene blue and xanthine oxidase-methylene blue systems.
The present study has also revealed that in the absence of methylene blue 0; is the key activator of indoleamine 2,3dioxygenase. Even in the presence of methylene blue, superoxide 'anion still contributes to about 50% of the dioxygenase activation at relatively low levels of xanthine oxidase (1.7 milliunits/ml) where less than 10% of the maximal activity is attained (Fig. 2B). At higher xanthine oxidase concentrations, OX-mediated activation diminishes to negligible extents. To explain these results, various OXand methylene blue-mediated reactions that are relevant to the present study are shown below, where MB represents methylene blue, L-MB leuco-methylene blue (2-electron-reduced MB), S-MB. a short-lived semimethylene blue radical (1-electron-reduced MB) (28)(29)(30), and ID0 indoleamine 2,3-dioxygenase. Without methylene blue, all of the electrons derived from a substrate of xanthine oxidase are used for the univalent and divalent reduction of O2 to generate OX and H202 (i) (31)(32)(33). Thus, 0; is the sole activator of the dioxygenase (u). When methylene blue (25 PM) is added, the dye and O2 compete for electrons from xanthine oxidase. Methylene blue can be reduced to leucomethylene blue most likely involving semi-methylene blue (ii). The lack of the inhibitory effects of superoxide dismutase at above 10 milliunits/ml xanthine oxidase (Fig. 3A ) strongly suggests that methylene blue at 25 p~ concentration might well predominate over O2 at any oxidase level in accepting electrons that would be used for the univalent reduction of Oz in the absence of the dye. An analogous case was reported in a past study by Muraoka et al. (34) where menadione (33 PM) dominates over O2 as an electron carrier from xanthine oxidase to ferricytochrome c. When xanthine oxidase concentrations are low, i.e. fewer electrons are available, small amounts of leuco-methylene blue and semi-methylene blue are generated. The reduced dye thus formed can donate electrons either to the ferric dioxygenase (70 nM) (iiia, iiib) or to Oz (250 p~) (iua, iub). The latter leads to reaction u. Apparently, the reactions iiia, iiib and iua, iub or u have comparable rates under these conditions. At higher xanthine oxidase levels, leuco-methylene blue and semi-methylene blue are gener-  (35) where ferricytochrome c was reduced totally by reduced methylene blue (1-10 PM) rather than by the OX that was generated by the autoxidation of the reduced dye under aerobic conditions. Although the actual OX concentrations under the various conditions used in this study can not easily be determined because of its quite unstable nature, i.e. disproportionation reaction ui, and although no attempts to determine 0; generation rates in the xanthine oxidase-hypoxanthine system have been done in this study, it is possible to estimate these values. Based on a previous study by Fridovich on the percent 0; generation per total electrons donated from xanthine (2 electrons/xanthine) and its pH dependence (31), and considering that 1 unit of xanthine oxidase using hypoxanthine as substrate (the unit used in this study) corresponds to approximately 2 units with xanthine being used as substrate (36) Leuco-methylene blue is shown to be able to reduce ferric indoleamine 2,3-dioxygenase (reaction iiia) to the extents of 25-40% under the anaerobic conditions employed, using -5 PM enzyme and 25-125 FM dye (Figs. 5 and 6). The incomplete reduction is most likely due to a relatively low oxidationreduction potential of the dioxygenase as compared with that of methylene blue (E'o = 0.011 V at pH 7.0 and 30 "C (38)).
The present results suggest that the E'o value for a ferricferrous pair of indoleamine 2,3-dioxygenase is considerably lower than that of sperm whale myoglobin (E'o = 0.05 V (39)) but higher than that of methyl viologen (E'o = -0.44 V (38)).
The significant increases in the extent of the dioxygenase reduction by the additions of ~-T r p and the heme ligands are apparently due to a shift of the following equilibrium reactions to the right. IDO(II1) + 1/2 leuco-MB -IDO(I1) + 1/2 MB IDO(I1) + L-TW C , L -T~~-I D O ( I I ) IDO(I1) + ligand -IDO(I1)-ligand ~-T r p has about 100 times higher affinity for the ferrous dioxygenase than for the ferric enzyme (27). CO and n-butyl isocyanide bind exclusively to the ferrous heme of the dioxygenase; the latter can bind to the ferric enzyme with a very low affinity (& = -W 3 M).3 Similar effects of o2 as those for these heme ligands can be expected. Even though the O2 adduct of the ferrous enzyme is autoxidizable, especially in the presence of substrate (15, 18), and even though leucomethylene blue is readily reoxidized by 0 2 to methylene blue (reactions iua and uiii), under catalytic reaction conditions, electrons are continuously supplied from available sources such as ascorbate or xanthine oxidase to maintain a steady state concentration of leuco-methylene blue at a certain level.
Based on the present results and interpretations, all likely pathways for the reductive activation of indoleamine 2,3dioxygenase by xanthine oxidase-hypoxanthine and by ascorbate are schematically summarized in Fig. 7. The routes marked el, e2, e3 and e4 represent direct electron transfer pathways that do not involve OH. The pathways for the reduction of O2 by leuco-methylene blue to generate 0; that may contribute to the activation of the dioxygenase under certain conditions are shown by dashed arrows. For the maximal enzyme activity, the pathways indicated by e3 and e4 are considered to be the major activation routes. The presence of  (Trp-IDO-Fe2+.02). The upper half above the middle horizontal arrow is for the xanthine oxidase (X0)-hypoxanthine-methylene blue (MB) co-factor system and the lower half for the ascorbatemethylene blue co-factor system. The arrows intercepted with two parallel bars represent superoxide dismutase (SOD)-inhibitable pathways. In this scheme, reduced methylene blue (MBM) rather than leuco-methylene blue is used for the reduced form(s) of methylene blue (cf. MBod, the oxidized form) in order to include the possible involvement of a short-lived, 1-electron-reduced form of methylene blue, ie. semi-methylene blue as a primary product or as a direct reductant in the methylene blue-mediated oxidation and reduction reaction pathways. Trp + 0, which is placed in a rectangle at the final step of the activation process is common to both the xanthine oxidase-methylene blue and ascorbate-methylene blue co-factor systems.
Trp and 0 2 will help shift the ferric form c, ferrous form equilibrium to the right.
The present study has not answered the questions as to ( a ) why 01 alone, even at its sufficiently high concentrations (-5 X M) cannot maintain the linearity of the catalytic reaction and ( b ) why methylene blue together with 0; can restore the activity of the "OH-inactivated" enzyme. Superoxide anion can reduce methylene blue according to reaction uii with a relatively high second order rate constant ( k = lo5-lo6 M" s" at pH 7.0 and 25 "C).' Hence, the leuco-methylene blue thus generated may reactivate the "0;-inactivated" enzyme. The cause and mechanism of the reversible "0;-inactivation" of the dioxygenase remain to be answered. The reduction of the ferric enzyme with leuco-methylene blue is so slow (Figs. 5 and 6) as compared with the catalytic rate constant (-2 s?). T o explain the reason for this, the inclusion of the semi-methylene blue radical as a potent reductant of the ferric dioxygenase (cf. reactions iiib) might be necessary, since semi-methylene blue is a more reactive and stronger reductant than leuco-methylene blue (29). Semi-methylene blue might be preferably generated under aerobic rather than the anaerobic conditions employed in the present study. In fact, in the ascorbic acid-methylene blue co-factor system under air, when the reduction of the dioxygenase is monitored by trapping the reduced enzyme with CO to generate the stable ferrous-CO enzyme, much faster reduction rates ( tLh: 25 and 70 s with and without ~-T r p , respectively) are ~b t a i n e d .~ Under similar conditions, superoxide dismutase has no inhibitory effect on the catalytic reaction of indoleamine 2,3dioxygenase (see above).
Xanthine oxidase is one of the likely candidates for the physiological electron donors to indoleamine 2,3-dioxygenase either uia O2 or other carriers, since the oxidase has been shown to be abundant in the small intestine and lung (40,41) where relatively high dioxygenase activity is found in rabbit (3,7), mice (9), and rats (10). However, it would be unrealistic to assume that over lo-' M steady state concentrations of 0; are available (42,43) in the cytosol of the tissues where the dioxygenase is located (5-7). As indicated in this study, low levels of 0; (5 X M) can directly activate the dioxygenase up to about 10% of its maximal activity. Since K , values for L-TT (K,,, = M at pH 7-7.5 at 25 "c) (6, 27) and for 0; (K, = M at pH 8.0 and 24 "C) (14) are relatively low, 0; may be utilized for ~-T r p metabolism in vivo by the dioxygenase, assuming that ( a ) the enzyme can compete with superoxide dismutase (42) for 0, or that ( b ) the dioxygenase normally operates at low percent levels of its maximal activity under physiological conditions. If, however, we expect nearmaximal activity of the dioxygenase in tissues, some physiological electron carrier(s) (44,45) between the donor(s) and the dioxygenase might exist. Such an activation process may not require OH. In either event, the physiological significance of the uniquely high reactivity of ferric indoleamine 2,3dioxygenase toward OH remains an intriguing question.