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THE JOURNALBIOLOGICAL OF CHEMISTRY 0 1985 by The American Society of Biological Chemiats, Inc.

Vol. 260, No. 3, Issue of February 10, pp. 1400-1406 1985 Printed in ~T.s.A.

Infrared Spectraof Carbon Monoxide Complexes of Indoleamine 2,3-Dioxygenase and L-Tryptophan 2,3=Dioxygenases
EFFECTS OF SUBSTRATES ON THE CO-STRETCHING FREQUENCIES*
(Received for publication, July 12, 1984)

Kiyoshi UchidalQ,Hiroshi Bandown, Ryu MakinoS, Kazuo SakaguchiS, Tetsutaro IizukaS, and Yuzuru IshimuraSII
From the $Department of Biochemistry, School of Medicine, Keio University, 35 Shimnomuchi, Shinjuku-ku, Tokyo 160 and the llDivision of Atmospheric Environment Division, the National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited twoCO stretch bands at 1963 and 1933 cm with half-band widths (Avllz) of both approximately 16 cm. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm- with the A v l ~ ~ 15cm-. Carbonmonoxy Lof tryptophan 2,3-dioxygenase of Pseudomonas acidouoralzs in theabsence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965and 1958cm- (Avllz for the fused band; 26 cm), which was converted into sharp single bandat 1968 a cm (Avllz; 10 cm) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver Ltryptophan 2,3-d1oxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm. By the addition of L-tryptophan, the spectrum changed into that with distinct bands two at 1972 and 1920cm (Avlla; 6 and 13 cm, respectively). These spectra were insensitive pH in a range to to where the enzymes were not denatured (neutral near pH 9). The infrared spectraof the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and a-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturatingamount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.

Indoleamine 2,3-dioxygenase from rabbit small intestine is an enzyme composed of a single polypeptide chain with a molecular weight of 42,000 containing 1mol of protoheme IX, while L-tryptophan 2,3-dioxygenases (EC 1.13.1.12) from both rat liver and Pseudomonas acidovorans (ATCC 11299b) are
* This work was supported, in part, by Grants in Aid for Scientific from the Ministry of 579110, and 58480450 Research 57480430, Education, Science and Culture, Japan, and by Grant in Aid of New Drug Development from the Ministry of Health and Welfare, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Pharmaceutical Institute, Tohoku University, Sendai 980, Japan. I( To whom all correspondence should be addressed.

tetrameric proteins containing 2 mol of the same heme with molecular weights of 167,000 and 122,000, respectively (1-3). Both kinds of enzymes catalyze the oxygenative conversion of L-tryptophan to L-formylkynurenine at the expense of 1 mol of molecular oxygen but differ in their substrate specificities; L-tryptophan 2,3-dioxygenase utilizes L-tryptophan and molecular oxygen almost exclusively astheir substrates, while indoleamine 2,3-dioxygenase can utilize a series of tryptophan analogues including L- and D-tryptophan and 5-hydroxytryptophan, and both 0 2 and 0; as the oxygenating agent (1-3). Among many hemoproteins, these enzymes are so far only dioxygenases whichhave been sufficiently purified and characterized to allow physicochemical investigation. In the present study, we examined infrared stretch bands for carbon monoxide (CO) bound to theferrous heme in these dioxygenases with special emphasis on the effects of L-tryptophan and other substrate analogues on the CO stretching frequencies. The results serve as the first demonstration of the CO stretch bands for the dioxygenase type of hemoproteins. Being in a region between 1974 and 1902 cm, their CO stretching frequencies lie in the same wave number region to those of other hemoproteins hitherto studied including oxygen carriers, cytochrome oxidase, peroxidases, and cytochrome P-450 (4, 5). However, both frequencies and shapes of their CO stretch bands were remarkably altered by the binding of their specific substrates, inhibitors and/or effectors with the enzymes. Amongthem, the binding of a substrateat the catalytic site gave most striking effects on the spectra, while effector binding at the regulatory site resulted in only small changes. Such findings are interpretedto mean that the catalytic binding site for L-tryptophan exists in a close proximity to the CO binding site (heme) and that the bound Ltryptophan at the site affects configuration of the CO-heme complex presumably through steric interaction. On the other hand, the binding at theregulatory site which may beremote from the heme site affects the CO stretch mode only slightly via the conformational changes of the protein.
MATERIALS AND METHODS Both indoleamine 2,3-dioxygenase fromrabbit small intestine and L-tryptophan 2,3-&oxygenase from P. acidouoruns (ATCC 11299b)

A recent report by Watanabe et al. (39) by using a sensitive assay methods with radioisotopes revealed the ratio of activities toward Dand L-tryptophan were 0.07 and less than 0.01 for the rat liver and Pseudomonas enzyme, respectively. The ratio was, however, shown to be species variable with the highest value of 0.67 for the ox liver enzyme. Cytochrome c oxidase is known to exhibit a CO dioxygenase activity (40, 41).However, the enzyme is usually regarded as an oxidase but not a dioxygenase.

1400

IR Spectra of CO Complexes of Heme-containing Dioxygenases


were purified as described previously (6-8). L-Tryptophan 2,3-dioxygenase from livers of male Wistar rats was prepared also according to the described method (8) except for the following modifications. The enzyme preparation from the Ultrogel AcA 34 chromatography in Ref. 8 was subjected to an affinity column with Sepharose 4B conjugated with L-tryptophan to remove small amounts of soluble btype hemoproteins in the liver preparation. The details for the preparation and use of the affinity column will be described elsewhere? Purities and specific activities of these enzyme preparations were comparable to those described in the preceding papers (6-8). Contamination of other hemoproteins such as hemoglobin, mitochondrial and microsomal cytochromes, and other b-type hemoproteins, catalase and peroxidases were not detected. Ferrous CO complexes of these enzymes were prepared by the addition of sodium dithionite to the solution of ferric enzyme equilibrated with CO a t 1 atom under anaerobic conditions. Ninety per cent-enriched 13C0was purchased from Merck Sharp and Dohme. L-Tryptophan, D-tryptophan, and skatole were purchased from Wako Pure Chemical Co. Ltd., Japan. 5-Hydroxy-~-tryptophan and a-methyl-DL-tryptophan were the products of Sigma. All other chemicals were of analytical grade from commercial sources and were used without further purification. Infrared spectra were measured with a Nicolet 7199 FT-IR spectrometer by single beam mode at room temperature. The use of FTIR allowed us quick measurements of the spectra of these unstable specimens a t low concentrations. The enzymes were unstable at room temperature, especially in their ferrous states in the presence of Ltryptophan (6); irreversible denaturation occurred rather rapidly to give a ferrous low-spin form which was catalytically inactive. Accumulation was 512 times with 1-cm-I resolution which took 15.5 min. Usually, changes in the spectra due to the denaturation of the enzymes were not observed within this time range. The detector employed was MCT (Mercury Cadmium Telluride) at 77 K. The cells were equipped with CaF2 windows in which the light path length was 0.1 mm. As the reference for the spectra of the CO complexes, either ferric or ferrous form of the enzyme was used giving essentially the same results with each other. Suitable concentrations of the enzymes for the infrared measurements were obtained by using an Amicon centriflow system. Otherdetailsare given under appropriate figure legends.
RESULTS
I

1401
I
1

1953

I
1900 WAVENUMEER(cm-')

2000

2000 1900 WAVENUMBER(cm'l)

1902
I

_I

FIG. 1 (left). Infrared spectra of carbonmonoxy indoleamine 2.3-dioxygenase in the presence and absence of L-tryptophan and its analogues. A , no addition; B, in the presence of 3 m LM tryptophan; C, in the presence of 1m skatole; D, in the presence of M 10 m D-tryptophan. The enzyme concentrations employed were 475, M 439, 363, and 356 p~ as protoheme for A , B, C, and D, respectively, in 0.1 M potassium phosphate buffer a t pH 7.3. The given spectra were all corrected to those with 475 pM as protoheme for comparison. In each case, the reference cuvette contained exactly the same components to those in the sample cuvette except that the CO complex of the enzyme was replaced by the ferric enzyme. FIG. 2 (right). Effects of skatole on the infrared spectrum of

27rC R. Makino, T. Iizuka, K. Sakaguchi, and Y. Ishimura, manuscript m2). Here k is the harmonic force constant, p is the reduced mass, in preparation. Pertinent portions of the work were presented at the and ml and m2 represent the masses of the two atoms in the diatomic 55th Annual Meeting of the Japan Biochemical Society, Tokyo, Oct. molecule. L-Tryptophan concentration was 3 m in both cases. M 11. 1982.

Figs. 1 and 2 show infrared spectra of ferrous carbon monoxide complex of indoleamine 2,3-dioxygenase under various indoleamine 2,3-dioxygenase in the presence of L-tryptophan. conditions in the region between 2050 and 1850 cm". A , in the presence of 0.4 m L-tryptophan; B, in the presence of both M M M In the absence of L-tryptophan and its analogues, the CO 0.4 m L-tryptophan and 1 m skatole; C, difference spectrum complex of the enzyme exhibited two bands at 1953 and 1933 between A and B ( A minus B). The enzyme concentration employed cm" (Fig. l ) These band positions were far remote from was 305 p~ as protoheme in 0.04 M potassium phosphate buffer, pH A. those of gaseous CO at 2145 cm" (9) and CO-hemocyanin, a 7.3. The ferrous form of the enzyme was used as reference. copper protein, at around 2050 cm" (lo), but were close to the positions of main CO stretching frequencies of CO-hemoTABLE I globin and CO-myoglobin (1951 and 1944 cm", respectively) Stretching frequencies of "CO and I3CO bound to the heme in three (4,5), andthose of synthetic heme models (1970cm") (9,11, dwxygenases and their comparison 12). When intensities of the two bands were compared, the band at 1953cm" had approximately twice as much intensity u(lzco) u(13c0) U ( ' T 0 Y as that at 1933 cm", while their band widths were roughly ern" the same (Avl12; 15 cm"). By the replacement of l2C0 with Indoleamine 2,3-dioxygenase 13C0,positions of both bands shifted to 1910 and 1890 cm", Without 1953 0.978 1910 respectively (Table I), where the magnitudes of the shift were substrate 0.978 1890 1933 in good agreements with the expected values for the vibraPseudomonas L-tryptophan 2,3tional frequency shift of a diatomic molecule upon changes in dioxygenase the mass number (4).Both of the observed bands could + L-tryptophanb 1968 0.978 1924 therefore by assigned to the stretching frequencies of CO Rat liver L-tryptophan 2,3-dioxygenase bound to the heme in the enzyme. Occurrence of such multiple Without substrate 1961 1916 0.977 CO stretch bands also been observed for the CO complexes has + L-tryptophan*0.977 1926 1972 of other monomeric hemoproteins such as myoglobin (13,14) 0.978 1878 1920 and cytochrome c peroxidase (15), and therefore is notunique "Expected value for U('~CO)/LJ('~CO) calculated to be 0.9778 was for the CO complex of this enzyme. On the other hand, CO complex of a denatured preparation the enzyme, which was based on the equation in Ref. 4,u = - where j~ = (mlmn)/(ml+ of ('), kp"

1402

IR Spectra of CO Complexes of Heme-containing Dwxygenases


addition of D-tryptophan up to the concentration of 10 m M at pH 7.5. It should be mentioned that D-tryptophan is neither the substrate nor an effector of the Pseudomonas L-tryptophan 2,3-dioxygenase (3, 18, 19). Effects of pH changes on the infrared spectra were not observed between 5.9 and 8.8. When effects of other tryptophan analogues on the spectra were examined, 5-hydroxy-~-tryptophan,5-flUorO-DL-tryptOphan, 6-fluoro-D~-tryptophan and gave similar spectral changes to that by L-tryptophan. The newly formed bands were all sharpgiving Avl/2 of 10 cm", although their positions were different from one another by2-3cm" (Fig. 3C and is Table 11).Among them, 6-fluoro-~~-tryptophana substrate for the enzyme, while 5-hydroxy-~-tryptophan and5-flUOrODL-tryptophan are competitive inhibitors of the reaction with respect to L-tryptophan (3). The Pseudomonas enzyme has been postulated to have twokinds of tryptophan binding sites, one the catalytic site and the other the regulatory site, and all of above analogues are considered to bind preferentially with the catalytic site (3, 18, 19). On the other hand, amethyl-DL-tryptophan, which is known to bind preferentially with the regulatory site (3,19), caused another typeof spectral change. Upon addition of a-methyl-DL-tryptophan (3 mM), the shoulder-like peak at 1958 cm" disappeared giving an apparently single maximum at 1967 cm-' with a broad Avl,z of 20 cm" (Fig. 30). No sharpening of the band was observed by increasing the concentration of a-methyl-DL-tryptophan up to 10 mM. It should be noted that intensities of the bands in Fig. 3 are not be compared with one another. The affinity to of ferrous enzyme for CO varies with the kind and amount of tryptophan analogues employed in the experiment, and hence the degrees of CO saturation are notthe same in each experiment. In the next experiment, we examined the effects oflow concentrations of L-tryptophan on the CO stretch bands, and found that a low concentration of L-tryptophan around the K,value (0.4 mM) gave a similar effect to that a saturating by amount of a-methyl-DL-tryptophan (Fig. 4). Because rather of poor base-lines for spectra, we were not able to carry out a precise curve analysis, which is neccesary to draw a definite conclusion on the similarity or dissimilarity of the spectra. Nevertheless, only slight differences were noted between the M spectra with a-methyl-DL-tryptophan (Fig. 4A) and 0.4 m L-tryptophan (Fig. 4B) and in the difference spectrum between them (Fig. 4C). The results were reproducible with several batches of enzyme preparation. On the other hand, when a dilute concentration of L-tryptophan was added to the CO complex in the presence of a-methyl-DL-tryptophan, almost identical spectrum to that with the saturating level of L-tryptophan was obtained (data not shown). These results thus indicated that the binding of L-tryptophan or its analogues to the catalytic site resulted in the formation of a sharp band at around 1968 cm" but the binding to the regulatory site caused only a decrease in the intensity of the band at 1958 cm". Furthermore, the observed effects of high and low concentrations of L-tryptophan in the presence and absence of a-methyl-DL-tryptophan suggested that L-tryptophan first binds to the regulatory site, facilitating its binding to the catalytic site. Fig. 5 shows infrared spectra of CO in the ferrous carbon monoxide complex of rat liver L-tryptophan 2,3-dioxygenase both in the presence and absence of L-tryptophan at pH 7.5. With the employed concentration of the enzyme (0.22 m as M heme), the spectrum showed an unresolved very broad peak around 1961 cm" in the absence of L-tryptophan (Fig. 5A). Since the liver enzyme is unstable particularly in the absence of L-tryptophan (20), we were not able to get more concen-

obtained by standing the ferric enzyme (300 PM) at pH 7.3 and room temperature for overnight, showed a single band at 1966 cm" with a half-band width of cm". 25 Thus, the presence of two bands in the infrared spectrum is not due to the contamination of a denatured species but is an inherent nature of the CO complex of indoleamine 2,3-dioxygenase. It remains to be elucidated, however, whether the multiple bands with this enzyme are due to multiple conformers of one CO binding site as in myoglobin (13, 14) or ascribable to more than one species of the binding sitesas in cytochrome c peroxidase (15). When L-tryptophan was added to the CO complex of indoleamine 2,3-dioxygenase, a new band at 1902 cm" appeared with concomitant decreases in theintensities of the bands at 1953 and 1933 cm" (Fig. 2 A ) . The magnitude of such spectral changes increased as L-tryptophan concentration increased, resulting in an essentially single band with a half-band width of 15 cm" at the L-tryptophan concentration of 3 m (Fig. M 1B). In a similar manner, additions of 5-hydroxy-~-tryptophan resulted in an appearance of a new band at 1904 cm". It was also found that theinfrared spectraof the CO complex in the presence and absence of 3 m L-tryptophan were M uneffected by changing pH from 5.9 to 8.8, and by changing (pD) 7.3. Further the reaction medium from H20 to D20at pH changes of the pH were not feasible because of instability of the enzyme.On theother hand, additions of skatole, an effector which stimulates the binding of L-tryptophan to the enzyme: resulted in a decrease in the intensity of the band at 1933 cm", giving a less well-resolved spectrum with two peaks at 1950 and 1936 cm" (Fig. IC). When used in combination with L-tryptophan, skatole facilitated the L-tryptophan-dependent changes in the spectra asshown in Fig. 2 (A, B, and C). However, additions of D-tryptophan, another substrate for this enzyme (1, 16), caused no significant change in the spectra of the CO complex bothin the presence and absence of skatole (Fig. 1D). The reason for this phenomenon is not understood but certain possibilities will be discussed later. These findings were reproducible by repeating the experiments with different batches of enzyme preparations. Fig. 3 shows infrared spectra of ferrous CO complex of the Pseudomonas L-tryptophan 2,3-dioxygenase in the presence and absence of L-tryptophan or its analogues. The CO complex without supplement showed a main peak at 1965 cm" accompanying with a prominent shoulder at around 1958cm" (Fig. 3A).Upon addition of 3 mM L-tryptophan, the spectrum turned into that with a single intense band at 1968 cm" with a half-band width of 10 cm" (Fig. 3B). By changing "CO to 13C0,the single band at 1968 cm" shifted to1924cm" (Table I) and was therefore assigned as the stretching frequency of CO bound to theheme iron in theenzyme. It should be noted that the enzyme concentration employed for the experiment in Fig. 3A was three times of that in Fig. 3B. Because of an unusually low affinity of the enzyme for CO in theabsence of L-tryptophan (17), ahigher concentration of the enzyme had to be used to obtain a measurable concentration of the CO complex in the absence of L-tryptophan. On the other hand, the ferrous enzyme in the presence of a sufficient amount of L-tryptophan shows a higher affinity for CO, which is comparable to those of other hemoproteins such as hemoglobin and myoglobin (17). Thus, thedegrees of CO saturation were different from one another in the experiments in Fig.3. Essentially no change in the spectrum was observed upon
K. Uchida, R. Makino, T. Iizuka, T. Shimizu, Y. Ishimura, Y. Nozawa, and M. Hatano, manuscript in preparation. Pertinent portions of the work were presented at the 53rd Annual Meeting of the Japan Biochemical Society, Tokyo, Oct. 15, 1980.

IR Spectra of CO Complexes of Heme-containing Dioxygenases


1965
I

1403

1961

1967
1

1967
1

C
I
1967

1900 WAVENUMBER (Cm")

2000

2 000 1900 WAVENUMBER (cm")

2000 1900 WAVENUMBER (cm")

FIG.3 (left). Infrared spectra of carbonmonoxy L-tryptophan 2,3-dioxygenase from Pseudomonas in thepresence and absence of L-tryptophan and its analogues. A, no addition; B, in the presence of 3 m M
M D, M L-tryptophan; C, in the presence of 2 m 5-hydroxy-~-tryptophan; in the presence of 3 m a-methyl-DLp~ tryptophan. The enzyme concentrations employed for the measurements were 1180 for A and 385,401, 385 in terms of protoheme concentration for A, B , C, and D,respectively, in 0 1 M potassium phosphate buffer, pH 7.5. . The given spectra B, C , and D were, however,corrected to those with the concentration of 418 pM for comparison. The ferric form of enzyme was used as reference. FIG.4 (center). Effects of low concentration of L-tryptophan on the spectra of carbonmonoxy Ltryptophan 2,3-dioxygenase from Pseudomonas. A, in the presence of 0.5 m L-tryptophan; B , in the M presence of 3 m a-methyl-DL-tryptophan; C , the difference spectrum between A and B (A minus B). The enzyme M concentrations employed for the measurements were 376 and 385 p~ in terms of protoheme concentration for A and B, respectively, in 0 1 M potassium phosphate buffer, pH 7.5. Given spectra were, however,corrected to those . with the concentration of 418 pM for comparison. The ferrous form of enzyme was used as reference. FIG.5 (right). Infrared spectra of carbonmonoxy L-tryptophan 2,3-dioxygenase from liver in the presence and absence of L- and D-tryptophan. A, with "CO in the absence of L-tryptophan; B, with ' T O in M M the absence of L-tryptophan; C, in the presence of 3 m L-tryptophan with "CO; D,in the presence of 3 m Dtryptophan with "CO. The enzyme concentrations employed in the experiments were 219,295,201, 270 p~ in and terms of protoheme concentration for A, B, C , and D,respectively, in 0 1 M potassium phosphate buffer, pH 7.5. . Given spectra were, however, corrected to those with the concentration of 219 p~ for comparison. The ferric form of enzyme was used as reference.

trated solutions which might allow high enough resolution of the fine structure of the spectrum. However, the peak around 1961cm" shifted to 1916 cm" by changing "CO to 13C0(Fig. 5B), where the magnitude of the shift by 45 cm" agreed well with the expected value of 44 cm" from the calculation (4). On the other hand, the enzyme showed two sharp peaks at 1972 and 1920 cm" in the presence of an excess amount of L-tryptophan with the half-band widths of 6 and 13 cm", respectively (Fig. 5C). These peaks at 1972 and 1920 cm" shifted to 1920 and 1878 cm" by the use of 13C0(Table I). It was suggested, therefore, that the CO complex of liver Ltryptophan 2,3-dioxygenase has one or more stretch bands around 1961 cm", which shifted or split into the two peaks upon addition of L-tryptophan. Essentially the same results were obtained at pH 8.5. When effects of other tryptophan

analogues were tested, D-tryptophan (Fig. 5D) and 5-hydroxyL-tryptophan showed similar spectral changes to those described above, whereas tryptamine gave somewhat different changes. CO stretching frequencies of these heme-containing dioxygenases in the presence and absence of various tryptophan analogues were summarized and compared with those of other hemoproteins in Tables I1 and 111.
DISCUSSION

For all the hemoproteins studied to date, infrared stretch bands for their CO complexes werefound in a region between 1966 and 1905 cm-' with a half-band width from 4 to 33 cm" (Table 111). They were shown to be sensitive to the changes in the ligand environment as well as in the electronic structure

1404

IR Spectra of CO Complexes of Heme-containing Dioxygenases


similar half-band width. Upon replacement of ' T O with W O , they exhibited reasonable shifts in wave number (Table I). Furthermore, both frequencies and widths of the bands were remarkably changed by the binding of their specific substrates or effectors with each enzyme. Thus, the observed peaks in the infrared spectra have been assigned to the CO stretch bands for these enzymes. Besides, the coincidence of the CO stretching region for the dioxygenase to that for the other hemoproteins suggested that electronic structures of their CO complexes werenot greatly different from those of others. When effects of various substrates and their analogues on infrared spectra were compared, the changes were most prominent with the saturating level of L-tryptophan, the natural substrate for the enzymes. Number of peaks, their shapes and frequencies were all markedly affected by L-tryptophan. Certainsubstrate analogues, especially competitive inhibitors against L-tryptophan such as5-hydroxy-~-tryptophan had similar effects on the spectra. On the other hand, effectors such as a-methyl-DL-tryptophan and skatole, which are neither substrates nor competitive inhibitors of these enzymes caused smaller spectral changes which wereof different types from that by the saturating amount of L-tryptophan. In the case of the Pseudomonas enzyme complex, however,a similar spectral change to that evoked by an effector was produced by a dilute concentration of L-tryptophan. Furthermore, when used in combination with the effector, the dilute L-tryptophan exerted a strong effect on the spectrum which was indistinguishable from that by the saturating amount of L-tryptophan. These results together with the previous findings of Feigelson and his co-workers (3, 19) that there exist both catalytic and regulatory binding sites for L-tryptophan in the enzyme indicated that the binding of asubstrate to the catalytic site evokes prominent changes in the spectra, while the binding at the regulatory site gives only small changes. Such an interpretation seems to be also valid for the case of

of the heme-ligand complex (4,5). In the present study, each dioxygenases had one or two absorption peaks in the same frequency region to those of other CO hemoproteins with a
TABLE I1 Effects of L-tryptophan and its analogues on the CO stretching frequencies of the dioxygenases Enzymes and additions uCO ( A u ~
em"

Indoleamine 2,3-dioxygenase None L-Tryptophan (3 mM)O D-Tryptophan (10 mM) 5-Hydroxy-~-tryptophan (10 mM) Skatole (1 mM) Pseudomonas L-tryptophan 2,3-dioxygenase None

1953 (15) 1902 (15) 1953 (15) 1953 (15) 1950 1936 (-18) 1965 & 1958 (-25) 1968 (10) 1970 (10)

1933 (15) 1933 (-15) 1934 1904 (-15) (-15) (15)

L-Tryptophan (3-10 mM) 5-Hydroxy-~-tryptophan (1-10 mM) 5-Fluoro-~~-tryptophan (3 1966 (10) mM) 6-Fluoro-~~-tryptophan (3 1968 (10) mM) a-Methyl-DL-tryptophan 1967 (20) (3 mM) Rat liver L-tryptophan 2,3dioxygenase 1961 (>20) None 1972 (6) 1920 L-Tryptophan (3 mM) 1969 (7) 1925 D-Tryptophan (3 mM) 1973 (7) 1928 5-Hydroxy-~-tryptophan (3 mM)b 1974 1952 (-10) (10) Tryptamine (3 mM) ~.
a

Trace peaks were present at 1953 and 1933 cm". A trace peak was observable at 1952 cm".

TABLE 111
C - 0 stretching frequencies and their half-band width for the carbonyl complexes of various hemoproteins Hemoproteins uC0 ( AuJ References
cm"

Indoleamine 2,3-&oxygenase" L-Tryptophan 2,3-dioxygenase" Pseudomonas Rat liver Hb A (human) Mb (sperm whale) Leghemoglobin a (soybean) Neutral pH Acidic pH Horseradish peroxidase A2 pH 9.0 pH 5.0 C pH 11.0 pH 7.1 Cytochrome P-450cam No addition + D-Camphor P-420 Chloroperoxidase PH 3 PH 6 Cytochrome c oxidase Bovine heart Cytochrome c peroxidase pH 6.4 pH 8.5 In the absence of substrates, inhibitors or effectors. Two bands were fused; see text. A small peak.

1953 (15), 1933 (15) 1965 and 1958 (25)b 1961 (>20) 1951 (8) 1944 (12), 1931 1947.5 (6) 1957 (>lo)

This paper This paper 21) (4, 599) (11, (22) (23)

1938 ( l l ) , 1925 (10) 1938 (-ll), 1906 (19) 1933 (16), 1929 (shoulder) 1933 (12), 1905 (17) 1963 (11-12), 1942 (19-21Y 1940 (13) 1966 (-23) (24) 1942 (-30) 1963.5 (4) 1922 (12.5) 1948 (33)

IR Spectra of CO Complexes of Heme-containing Dioxygenases

1405

Most of our present results are well explained in terms of indoleamine 2,3-dioxygenase. We recently obtained evidence that the enzyme has also a regulatory binding site for L- above postulations. However, we could not explain why the tryptophan besides the catalytic binding site; additions of addition of D-tryptophan had no significant effect on the CO certain indole derivatives such as skatole and indole stimu- stretch bands of indoleamine 2,3-dioxygenase. Similar inertlated the catalytic activity of the enzyme, when a low concen- ness of D-tryptophan on the CO complex has also been noticed tration of D-tryptophan was used as thesubstrate. The details in our recent studies on magnetic and naturalcircular dichrowill be described el~ewhere.~ From these findings and results ism spectra of this enzyme (7). These findings were in contrast a herein described, we suggest that the prominent changes in with those of L-tryptophan which always givesmarked effect the infrared spectrum of indoleamine 2,3-dioxygenase is also on the spectrum irrespective of the valence, spin, and ligand binding states of the enzyme. Being a substrate,D-tryptophan caused by the substratebinding to thecatalytic site. Question then arises as to themechanism by which bound should bind to the catalytic site during the catalysis to form 2 substrate at the catalytic siteaffects the CO stretch bands. In a ternary complex of 0 and D-tryptophan with the enzyme, this regard, a possible correlation between the steric structure as has been observed with L-tryptophan as substrate (35,36). of CO-heme adduct and CO stretching frequency is notewor- Then, the formation of aternary complex ofCO and Dthy. It has been shown by using model systems that Fe-C-0 tryptophan with the enzyme can also be expected. Neverthebond is essentially linear in the absence of steric crowding less, we could not obtain any evidence for the binding of D(27,28) while the same bond in hemoproteins is bent or tilted tryptophan with the CO complex of indoleamine 2,3-dioxydue to the steric crowding around the bound CO (29, 30). genase as stated above. In this regard, the following possibilWhen CO stretching frequencies were measured, synthetic ities maybe considered ( a ) D-tryptophan can bind to the heme models such as diethylprotoporphyrinatoiron-l-meth- catalytic site but does not affect the configuration of Fe-C-0 ylimidazole and picket fence iron porphyrin-l-methylimida- nor cause the changes in steric crowding around the heme zole, where Fe-C-0 bonds are presumably linear, showed the because of its D-configuration, and ( b ) D-tryptophan is pracstretch bands around 1970 cm" (12), while the values for tically unable to bind with the CO complex because of its low affinity toward the complex. In this connection, a difference ordinary hemoproteins were always lower than 1970 cm" (Table 111). Such lower CO stretching frequencies in hemo- between the structures of CO and O2 adducts of heme iron proteins have been ascribed to the*-back donation from iron has been noted. It has been shown that Fe-0-0 bond is bent even without steric hindrance (37, 38), d-orbital to CO **-orbital (31-33), of which extent isdepend- inherently ent on the steric structureof Fe-C-0 (34). Then, theobserved whereas that of Fe-C-0 is essentially linear in the absence of shifts in the CO stretching frequencies of the dioxygenases steric pressure (27, 28). Experiments to discriminate these upon substrate binding can also be correlated with the changes possibilities are now under progress. Finally, detection of at least two CO stretch bands for in steric structure of the CO-heme adduct caused by the substrate binding at thecatalytic site. In these dioxygenases, Pseudomonas enzyme without L-tryptophan and also for rat the catalytic binding site for L-tryptophan is considered to liver enzyme with an excess amount of L-tryptophan might exist in a close proximity to the heme iron; the bound L- suggest the nonequivalency of the hemes in these enzymes tryptophan and oxygen at the heme must react with each under the experimental conditions. Both enzymes have two other to yield the reaction product. It is not surprising there- protohemes/mole of enzymes which are tetramers. Indoleamfore that configuration of the CO-heme adduct is altered by ine 2,3-dioxygenase, which is a monomer with a single heme, the steric pressure of the bound substrate itself or by that of also showed two bands in the absence of L-tryptophan indiof amino acid residue(s) of the protein which is forced to move cating certain kinds heterogeneity in itsheme-CO structure under the conditions. Possible effects of such nonequivalency by the substratebinding to thecatalytic site. The observed insensitivity of the CO stretch bands against or heterogeneity on the other physicochemical properties, pH is worthy of note. Since hydrogen bonding is known to be catalytic and ligand binding activities of these enzymes resensitive to pH, the finding may suggest that changes in main to be elucidated. hydrogen bond network around CO-heme adduct do not play Acknowledgments-We wish to thank Dr. K. Kuratsuka and his a crucial role in the changes of CO stretching frequency. Effects of other electronic factors such as electron density associates, National Institute of Health, Japan, for supplying us fresh distribution in the iron-porphyrin may not be so large as rabbit intestine. We are also indebted to Y. Tanizaki and A. 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