J. Biol. Chem.-1985-Uchida-1400-6

8/3/2019 J. Biol. Chem.-1985-Uchida-1400-6

1/7

THE OURNALF BIOLOGICALCHEMISTRY0 1985by The American Societyof Biological Chemiats, Inc. Vol. 260, No. 3, Issue of February 10,pp. 1400-1406 1985Printed in ~T.s.A.

Infrared Spectraof Carbon Monoxide Complexes of Indoleamine2,3-Dioxygenase and L-Tryptophan 2,3=DioxygenasesEFFECTS OF SUBSTRATES ON THE CO-STRETCHING FREQUENCIES*

(Received for publication, July 12, 1984)

Kiyoshi UchidalQ,Hiroshi Bandown, Ryu MakinoS, KazuoakaguchiS, Tetsutaro IizukaS, andYuzuru IshimuraSIIFrom the $Department of Biochemistry, School of Medicine, Keio University, 35 Shimnomuchi , Shinjuku-ku, Tokyo 160 andthe llDivision of Atmospheric Environment Division, the National Institute for Environmental Studies, Yatabe,Tsukuba, Ibaraki 305,Japan

Carbonmonoxy indoleamine 2,3-dioxygenase fromrabbit small intestine exhibited twoCO stretch bandsat 1963 and 1933 cm with half-band widths (Avllz)of both approximately 1 6 cm. Upon addition of anexcess amount of L-tryptophan, the substrate , thepec-trum changed into that with an intenseingle band at1902cm- with the A v l ~ ~f 15 cm-. CarbonmonoxyL-tryptophan 2,3-dioxygenase of Pseudomonas acido-uoralzs in theabsence of L-tryptophan showed a fusedCO stretch band which consists of two components at1965and 1958cm- (Avllz for the used band; 26 cm),which was converted into sharp single bandat 1968cm (Avllz; 10 cm) upon addition of excess L-trypto-phan. On the other hand, CO complex of rat liver L-tryptophan 2,3-d1oxygenase in the absence of L-tryp-tophan gave a spectrum with a poorly defined peakaround 1961 cm. By the addition of L-tryptophan,the spectrum hanged into that withwo distinct bandsat 1972 and 1920cm (Av l la ;6 and 13 cm, respec-tively). These spectra were insensitive toH in a rangewhere the nzymes were not denatured (neutralo nearpH 9).The infrared spectraof the carbonmonoxy enzymeswere also affected by the addition of certain effectorssuch as skatole and a-methyl-DL-tryptophan, whichfacilitate the binding of L-tryptophan to the ca talyticsite of intestinal and Pseudomonas enzymes, respec-tively. However, the changes were of different typesfrom those by the saturatingamount of L-tryptophan.Possible mechanisms for these phenomena are dis-cussed in relation to the structure of the heme-COcomplex in these heme-containing dioxygenases.

Indoleamine 2,3-dioxygenase from rabbit small intestine isan enzyme composed of a single polypeptide chain with amolecular weight of 42,000 containing 1mol of protoheme IX ,while L-tryptophan 2,3-dioxygenases (EC 1.13.1.12) from bothrat liver and Pseudomonas acidovorans (ATCC 11299b) areResearch 57480430,579110, and 58480450 rom the Ministry of* This work was supported, in par t, by Gran ts in Aid for ScientificEducation, Science and Culture, Japan, and by Grant in Aid of NewDrug Development from the Ministry of Health and Welfare, Japan.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be herebymarked aduertisement in accordance with 18U.S.C. Section 1734solely 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 withmolecular weights of 167,000 and 122,000, respectively (1-3).Both kinds of enzymes catalyze the oxygenative conversionof L-tryptophan to L-formylkynurenine at the expense of 1mol of molecular oxygen but differ in their substrate specific-ities; L-tryptophan 2,3-dioxygenase utilizes L-tryptophan andmolecular oxygen almost exclusively as heir substrates,while indoleamine 2,3-dioxygenase can utilize a series of tryp-tophan analogues including L- and D-tryptophan and 5-hy-droxytryptophan, and both 0 2 and 0; as he oxygenatingagent (1-3). Among many hemoproteins, these enzymes areso far only dioxygenases whichhave been sufficiently purifiedand characterized to allow physicochemical investigation.In the present study, we examined infrared stretch bandsfor carbon monoxide (CO) bound to the errous heme in thesedioxygenases with special emphasis on the effects of L-tryp-tophan and other substrate analogues on the CO stretchingfrequencies. The results serve as the first demonstration ofthe CO stretch bands for the dioxygenase type of hemopro-teins. Being in a region between 1974 and 1902 cm, theirCO stretching frequencies lie in the same wave number regionto those of other hemoproteins hitherto studied includingoxygen carriers, cytochrome oxidase, peroxidases, and cyto-chrome P-450 (4, 5). However, both frequencies and shapesof their CO stretch bands were remarkably altered by thebinding of their specific substrates, inhibitors and/orffectorswith the enzymes. Among hem, the binding of a substrateatthe catalytic site gave most striking effects on the spectra,while effector binding at the regulatory site resulted in onlysmall changes. Such findings are interpreted o mean that thecatalytic binding site for L-tryptophan exists in a close prox-imity to the CO binding site (heme) and that the bound L-tryptophan a t the site affects configuration of the CO-hemecomplex presumably through steric interaction. On the otherhand, the binding at the egulatory site which may be emotefrom the heme site affects the CO stretch mode only slightlyvia the conformational changes of the protein.

MATERIALS AND METHODSL-tryptophan 2,3-&oxygenase from P. acidouoruns (ATCC 11299b)Both indoleamine 2,3-dioxygenase from rabbit small intestine and

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

1400


2/7

I R Spectra of CO Complexesof Heme-containing Dioxygenaseswere purified as described previously (6-8). L-Tryptophan 2,3-dioxy-genase from livers of male Wistar rat s was prepared also accordingto the described method (8) except for the following modifications.Th e enzyme preparation from the Ultrogel AcA 34 chromatographyin Ref. 8 was subjected to an affinity column with Sepharose 4Bconjugated with L-tryptophan to remove small amounts of soluble b-type hemoproteins in t he liver preparation. Th e details for the prep-aration and use of the affinity column will be described elsewhere?Purit ies and specific activities of these enzyme preparations werecomparable to those described in the preceding papers (6-8). Contam-ination of other hemoproteins such as hemoglobin, mitochondrial andmicrosomal cytochromes, and other b-type hemoproteins, catalaseand peroxidases were not detected. Ferrous CO complexes of theseenzymes were prepared by the addition of sodium dithionite to thesolution of ferric enzyme equilibrated with CO at 1 atom underanaerobic conditions. Ninety per cent-enriched 13C0 was purchasedfrom Merck Sharp and Dohme. L-Tryptophan, D-tryptophan, andskatole were purchased from Wako Pure Chemical Co. Ltd., Japan.5-Hydroxy-~-tryptophan and-methyl-DL-tryptophan were the prod-ucts of Sigma. All other chemicals were of analytical grade fromcommercial sources and were used without further purification.Infrared spectra were measured with a Nicolet 7199 FT-IR spec-trometer by single beam mode at room temperature. The use of FT-IR allowed us quick measurements of the spectra of these unstablespecimens a t low concentrations. Th e enzymes were unstable a t roomtemperature, especially in their ferrous states in the presence of L-tryptophan (6); irreversible denaturation occurred rather rapidly togive a ferrous low-spin form which was catalytically inactive. Accu-mulation was 512 times with 1-cm-I resolution which took 15.5 min.Usually, changes in the spectra due to the enaturation of the enzymeswere not observed within this time range. The detector employed wasMCT (Mercury Cadmium Telluride) at 77 K. The cells were equippedwith CaF2 windows in which t he light path length was 0.1 mm. Asthe reference for the spectra of the CO complexes, either ferric orferrous form of the enzyme was used giving essentially the sameresults with each other. Suitable concentrations of the enzymes forthe infrared measurements were obtained by using an Amicon cen-triflow system. Otherdetailsare given under appropriate figurelegends.

RESULTSFigs. 1 and 2 show infrared spectra of ferrous carbon mon-

oxide complex of indoleamine 2,3-dioxygenase under variousconditions in the region between 2050 and 1850 cm".In the absence of L-tryptophan and its analogues, the COcomplex of the enzyme exhibited two bands at 1953 and 1933cm" (Fig. lA).These band positions were far remote fromthose of gaseous CO at 2145 cm" (9) and CO-hemocyanin, acopper protein, at around 2050 cm" (lo) , but were close tothe positions of main CO stretching frequencies of CO-hemo-globin and CO-myoglobin (1951 and 1944 cm", respectively)(4,5), and hose of synthetic heme models (1970cm") (9,11,12) . When intensities of the two bands were compared, theband at 1953 cm" had approximately twice as much intensityas that at 1933 cm", while their band widths were roughlythe same (Avl12; 15 cm"). By the replacement of l2C0 with13C0,positions of both bands shifted to 1910 and 1890 cm",respectively (Table I) , where the magnitudes of the shift werein good agreements with the expected values for the vibra-tional frequency shift of a diatomic molecule upon changes inthe mass number (4).Both of the observed bands couldtherefore by assigned to the stretching frequencies of CObound to the eme in the enzyme. Occurrence of such multipleCO stretch bands as also been observed for the CO complexesof other monomeric hemoproteins such as myoglobin (13,14)and cytochrome c peroxidase (15), and herefore is notuniquefor the CO complex of this enzyme. On the other hand, COcomplex of a denatured preparation f the enzyme, which was

R. Makino, T. Iizuka, K. Sakaguchi, and Y. Ishimura, manuscriptin preparation. Pertinent portions of the work were presented at th e55th Annual Meeting of the Japan Biochemical Society, Tokyo, Oct.11.1982.

2000 1900WAVENUMEER(cm-')L

1401I I 11953

r

I1902I I I2000 1900 _IWAVENUMBER(cm'l)

FIG. 1 left) . Infrared spectra of carbonmonoxy indoleamine2.3-dioxygenase in the presence and absence of L-tryptophanand its analogues. A , no addition; B , in the presence of 3 mM L-tryptophan; C, in the presence of 1mM skatole; D, in the presence of10 mM D-tryptophan. The enzyme concentrations employed were 475,439, 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 spectrawere all corrected to those with 475 pM as protoheme for comparison.In each case, the reference cuvette contained exactly the same com-ponents to those in the sample cuvette except tha t the CO complexof the enzyme was replaced by the ferric enzyme.FIG. 2 (right). Effects of skatole on he infrared spectrum ofindoleamine 2,3-dioxygenase in the presence of L-tryptophan.A , in the presence of 0.4 mM L-tryptophan; B , in the presence of both0.4 mM L-tryptophan and 1 mM skatole; C, difference spectrumbetween A and B ( A minus B ) . The enzyme concentration employedwas 305 p~ as protoheme in 0.04 M potassium phosphate buffer, pH7.3. The ferrous form of the enzyme was used as reference.

TABLEStretch ing frequencies of "CO and I3CO bound to the heme in threedwxygenases and their comparison

u(lzco) u(13c0) U ( ' T 0 Yern"

Indoleamine 2,3-dioxygenaseWithoutubstrate 1953 1910.9781933890.978Pseudomonas L-tryptophan 2,3-dioxygenase+ L-tryptophanb 1968 1924.978Rat liver L-tryptophan 2,3-di-

Without substrate 1961 1916 0.977+ L-tryptophan* 1972926.9771920878.978

"Expected value for U('~CO)/LJ('~CO)as calculated to be 0.9778based on the equation in Ref. 4, =-k'p)", where j~ = (mlmn)/(ml+m2). Here k is the harmonic force constant, p is the reduced mass,and ml and m2 represent the masses of the two atoms in the diatomicmolecule.

oxygenase

27rC

L-Tryptophan concentration was 3 mM in both cases.


3/7

1402 IR Spectra of CO Complexes of Heme-containing Dwxygenasesobtained by standing the ferric enzyme (300 PM ) at pH 7.3and room temperature for overnight, showed a single band at1966 cm" with a half-band width of25cm". Thus, thepresence of two bands in the infrared spectrum is not due tothe contamination of a denatured species but is an inherentnature of the CO complex of indoleamine 2,3-dioxygenase. Itremains to be elucidated, however, whether the multiple bandswith this enzyme are due to multiple conformers of one CObinding site as in myoglobin (13, 14) or ascribable to morethan one species of the binding sitesas in cytochrome cperoxidase (15).When L-tryptophan was added to the CO complex of in-doleamine 2,3-dioxygenase, a new band at 1902 cm" appearedwith concomitant decreases in the ntensities of the bands at1953 and 1933 cm" (Fig. 2 A ) . The magnitude of such spectralchanges increased as L-tryptophan concentration increased,resulting in an essentially single band with a half-band widthof 15 cm" at t he L-tryptophan concentration of 3 mM (Fig.1B). In a similar manner, additions of 5-hydroxy-~-trypto-phan resulted in an appearance of a new band at 1904 cm".It was also found that the nfrared spectraof the CO complexin the presence and absence of 3 mM L-tryptophan wereuneffected by changing pH from 5.9 to 8.8, and by changingthe reaction medium from H20 to D 2 0at pHpD) 7.3. Furtherchanges of the pH were not feasible because of instability ofthe enzyme.On theother hand, additions of skatole, aneffector which stimulates the binding of L-tryptophan to theenzyme: resulted in a decrease in the intensity of the bandat 1933 cm", giving a less well-resolved spectrum with twopeaks a t 1950 and 1936 cm" (Fig. IC). When used in combi-nation with L-tryptophan, skatole facilitated the L-trypto-phan-dependent changes in the spectra as shown in Fig. 2 (A,B, and C ) . However, additions of D-tryptophan, another sub-strate for this enzyme (1, 16), caused no significant change inthe spectra of the CO complex both n the presence andabsence of skatole (Fig. 1D). The eason for this phenomenonis not understood but certain possibilities will be discussedlater. These findings were reproducible by repeating the ex-periments with different batches of enzyme preparations.Fig. 3 shows infrared spectra of ferrous CO complex of thePseudomonas L-tryptophan 2,3-dioxygenase in the presenceand absence of L-tryptophan or its analogues. The CO com-plex without supplement showed a main peak at 1965 cm"accompanying with a prominent houlder at around 1958 cm"(Fig. 3A).Upon addition of 3 mM L-tryptophan, the spectrumturned into that with a single intense band at 1968 cm" witha half-band width of 10 cm" (Fig. 3B). By changing "CO to13C0, he single band at 1968 cm" shifted to1924 cm" (TableI) and was therefore assigned as the stretching frequency ofCO bound to theheme iron in theenzyme. It should be notedtha t the enzyme concentration employed for the experimentin Fig. 3A was three times of that in Fig. 3B. Because of anunusually low affinity of the enzyme for CO in theabsence ofL-tryptophan (17), ahigher concentration of the enzyme hadto be used to obtain a measurable concentration of the COcomplex in the absence of L-tryptophan. On the other hand,the ferrous enzyme in the presence of a sufficient amount ofL-tryptophan shows a higher affinity for CO, which is com-parable to those of other hemoproteins such as hemoglobinand myoglobin (17). Thus, thedegrees of CO saturation weredifferent 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.tions of the work were presented at the 53rd Annual Meeting of theNozawa, and M. Hatano, manuscript in preparation. Pertinent por-Japan Biochemical Society, Tokyo, Oct. 15 , 1980.

addition of D-tryptophan up to the concentration of 10 mMat pH 7.5. It should be mentioned tha t D-tryptophan is neitherthe substrate nor an effector of the Pseudomonas L-trypto-phan 2,3-dioxygenase (3, 18, 19). Effects of pH changes onthe infrared spectra were not observed between 5.9 and 8.8.When effects of other tryptophan analogues on the spectrawere examined, 5-hydroxy-~-tryptophan, -flUorO-DL-tryptO-phan,nd -fluoro-D~-tryptophan gave similar spectralchanges to that by L-tryptophan. The newly formed bandswere all sharp iving Avl/2 of 10 cm", although their positionswere different from one another by2-3cm" (Fig. 3C andTable 11).Among them, 6-fluo ro-~ ~-t ryptopha ns a substratefor the enzyme, while 5-hydroxy-~-tryptophan and -flUOrO-DL-tryptophan are competitive inhibitors of the reaction withrespect to L-tryptophan (3). The Pseudomonas enzyme hasbeen postulated to have twokinds of tryptophan binding sites,one the catalytic site and the other the regulatory site, andall of above analogues are considered to bind preferentiallywith the catalytic site (3, 18, 19). On the other hand, a-methyl-DL-tryptophan, which is known to bind preferentiallywith the regulatory site (3,19), aused another type f spectralchange. Upon addition of a-methyl-DL-tryptophan (3 mM),the shoulder-like peak at 1958 cm" disappeared giving anapparently single maximum at 1967 cm-' with a broad Avl,zof 20 cm" (Fig. 30 ). No sharpening of the band was observedby increasing the concentration of a-methyl-DL-tryptophanup to 10 mM. It should be noted that intensities of the bandsin Fig. 3 are noto be compared with one another. The affinityof ferrous enzyme for CO varies with the kind and amount oftryptophan analogues employed in the experiment, and hencethe degrees of CO saturation are not he same in each exper-iment.In the next experiment, we examined the effects of owconcentrations of L-tryptophan on the CO stretch bands, andfound that a low concentration of L-tryptophan around theK,value (0.4 mM) gave a similar effect to that y a saturatingamount of a-methyl-DL-tryptophan (Fig. 4). Becausef ratherpoor base-lines for spectra, we were not able to carry out aprecise curve analysis, which is neccesary to draw a definiteconclusion on the similarity or dissimilarity of the spectra.Nevertheless, only slight differences were noted between thespectra with a-methyl-DL-tryptophan (Fig. 4A) and 0.4 mML-tryptophan (Fig. 4B) and in the difference spectrum be-tween them (Fig. 4C). The results were reproducible withseveral batches of enzyme preparation. On the other hand,when a dilute concentration of L-tryptophan was added to theCO complex in the presence of a-methyl-DL-tryptophan, al-most identical spectrum to tha t with the saturating level ofL-tryptophan was obtained (data not shown). These resultsthus indicated tha t the binding of L-tryptophan or its ana-logues to the atalytic site resulted in the formation of a sharpband at around 1968 cm" but the binding to the regulatorysite caused only a decrease in the intensity of the band at1958 cm". Furthermore, the observed effects of high and lowconcentrations of L-tryptophan in the presence and absenceof a-methyl-DL-tryptophan suggested that L-tryptophan firstbinds to the regulatory site, facilitating its binding to thecatalytic site.Fig. 5 shows infrared spectra of CO in the ferrous carbonmonoxide complex of ra t liver L-tryptophan 2,3-dioxygenaseboth in the presence and absence of L-tryptophan at pH 7.5.With the employed concentration of the enzyme (0.22 mM asheme), the spectrum showed an unresolved very broad peakaround 1961 cm" in the absence of L-tryptophan (Fig. 5 A ) .Since the liver enzyme is unstable particularly in the absenceof L-tryptophan (20), we were not able to get more concen-


4/7

I R Spectra of CO Complexesof Heme-containing Dioxygenases 14031965

I

I 1967

J2000 1900WAVENUMBER (Cm")

I I I19671

19671

C I

I I I

I 1961

2000 1 9 0 0WAVENUMBER (cm")2000 1 9 0 0W A V E N U M B E R (cm")

FIG. (left). Infrared spectra of carbonmonoxy L-tryptophan 2,3-dioxygenase from Pseudomonasin thepresence and absence of L-tryptophan and it s analogues.A, no addition; B , in the presence of 3 mML-tryptophan; C , in the presence of 2 mM 5-hydroxy-~-tryptophan; , n the presence of 3 mM a-methyl-DL-tryptophan. The enzyme concentrations employed for the measurements were 1180 for A and 385,401, 385~ interms of protoheme concentration for A, B, C, and D, espectively, in 0.1 M potassium phosphate buffer, pH 7.5.Th e given spectra B , C , and D were, however, corrected to those with the concentration of 418pM for comparison.Th e ferric form of enzyme was used as reference.FIG. (center) . Effects of low concentration of L-tryptophan on the spectra of carbonmonoxy L-tryptophan 2,3-dioxygenase from Pseudomonas. A, in the presence of 0.5 mM L-tryptophan; B, in thepresence of 3 mM a-methyl-DL-tryptophan; C , the difference spectrum between A and B (A minus B ) . The enzymeconcentrations employed for the measurements were 376 and 385 p~ in terms of protoheme concentration for Aand B , respectively, in 0.1 M potassium phosphate buffer, pH 7.5.Given spectra were, however, corrected to thosewith the concentration of 418pM for comparison. The ferrous form of enzyme was used as reference.FIG. (r ight ) . Infrared spectra of carbonmonoxy L-tryptophan 2,3-dioxygenase from liver in thepresence and absence of L-and D-tryptophan. A, with "CO in the absence of L-tryptophan; B , with ' T O inthe absence of L-tryptophan; C, in the presence of 3 mM L-tryptophan with "CO; D, n the presence of 3 mM D-tryptophan with "CO. Th e enzyme concentrations employed in the experiments were 219,295,201,nd 270 p~ interms of protoheme concentration for A, B , C , and D, espectively, 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 formof enzyme was used as reference.trated solutions which might allow high enough resolution ofthe fine structure of the spectrum. However, the peak around1961 cm" shifted to 1916 cm" by changing "CO to 13C0 Fig.5B), where the magnitude of the shift by 45 cm" agreed wellwith the expected value of 44 cm" from the calculation (4).On the other hand, the enzyme showed two sharp peaks a t1972 and 1920 cm" in the presence of an excess amount ofL-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). Itwas suggested, therefore, that the CO complex of liver L-tryptophan 2,3-dioxygenase has one or more stretch bandsaround 1961 cm", which shifted or split into the two peaksupon addition of L-tryptophan. Essentially the same resultswere obtained a t pH 8.5. When effects of other tryptophan

analogues were tested, D-tryptophan (Fig. 5D) and 5-hydroxy-L-tryptophan showed similar spectral changes to those de-scribed above, whereas tryptamine gave somewhat differentchanges. CO stretching frequencies of these heme-containingdioxygenases in the presence and absence of various trypto-phan analogues were summarized and compared with thoseof other hemoproteins in Tables I1 and 111.

DISCUSSIONFor all the hemoproteins studied to date, infrared stretch

bands for their CO complexes were ound in a region between1966 and 1905 cm-' with a half-band width from 4 o 33 cm"(Table 111).They were shown to be sensitive to the changesin the ligand environment aswell as in the electronic structure


5/7

1404 IR Spectra of CO Complexes of Heme-containing Dioxygenasesof the heme-ligand complex (4,5). In theresent study, eachdioxygenases had one or two absorption peaks in the samefrequency region to those of other CO hemoproteins with a

TABLE1Effec ts of L-tr yptop han and its analogues on the CO stretchingfrequen cies of th e dioxygenasesEnzymes and additions uC O ( A u ~

Indoleamine 2,3-dioxygenaseNoneL-Tryptophan (3 mM)OD-Tryptophan (10 mM)5-Hydroxy-~-tryptophanSkatole (1 mM)(10 mM)Pseudomonas L-tryptophan2,3-dioxygenaseNoneL-Tryptophan (3-10 mM)5-Hydroxy-~-tryptophan

5-Fluoro-~~-tryptophan (3(1-10 mM)mM)mM)(3 mM)dioxygenase

6-Fluoro-~~-tryptophan (3a-Methyl-DL-tryptophan

Rat liver L-tryptophan 2,3-NoneL-Tryptophan (3 mM)D-Tryptophan (3 mM)5-Hydroxy-~-tryptophanTryptamine (3 mM)(3 mM)b

em"

1953 (15) 1933 (15)1902 (15)1953 (15) 1933 (-15)1953 (15) 1934-15) 904 (15)1950-18) 936 (-15)

1965& 1958(-25)1968 (10)1970 (10)

1966 (10)1968 (10)1967 (20)

1961 (>20)1972 (6) 19201969 (7) 19251973 (7) 19281974-10) 952 (10)

a Trace peaks were present a t 1953 and 1933 cm".~.

A trace peak was observable a t 1952 cm".

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 wereremarkably changed by the binding of their specific substratesor effectors with each enzyme. Thus, the observed peaks inthe infrared spectra have been assigned to the CO stretchbands for these enzymes. Besides, the coincidence of the COstretching region for the dioxygenase to that for the otherhemoproteins suggested that electronic structures of their COcomplexes were not greatly different from those of others.

When effects of various substrates and their analogues oninfrared spectrawere compared, the changes were most prom-inent with the saturating level of L-tryptophan, the naturalsubstrate for the enzymes. Number of peaks, their shapes andfrequencies were all markedly affected by L-tryptophan. Cer-tainsubstrate analogues, especially competitive inhibitorsagainst L-tryptophan such as5-hydroxy-~-tryptophan hadsimilar effects on the spectra. On the other hand, effectorssuch as a-methyl-DL-tryptophan and skatole, which are nei-ther substrates nor competitive inhibitors of these enzymescaused smaller spectral changes which wereof different typesfrom that by the saturating amount of L-tryptophan. In thecase of the Pseudomonas enzyme complex, however,a similarspectral change to that evoked by an effector was producedby a dilute concentration of L-tryptophan. Furthermore, whenused in combination with the effector, the dilute L-tryptophanexerted a strong effect on the spectrum which was indistin-guishable from tha t by the saturating amount of L-trypto-phan. These results together with the previous findings ofFeigelson and his co-workers (3 , 19) that there exist bothcatalytic and regulatory binding sites for L-tryptophan in theenzyme indicated that he binding of asubstrate to hecatalytic site evokes prominent changes in the spectra, whilethe binding at the regulatory site gives only small changes.Such an interpretation seems to be also valid for the case of

TABLE11C - 0 stretching frequencies and their half-band width for thearbonyl complexes of various hemoproteins

Hemoproteins uC0 (AuJ ReferencesIndoleamine 2,3-&oxygenase"L-Tryptophan 2,3-dioxygenase"PseudomonasRat liverHb A (human)Mb (sperm whale)Leghemoglobin a (soybean)Neutral pHAcidic pH

A2 pH 9.0pH 5.0C pH 11.0pH 7.1

No addition+ D-CamphorChloroperoxidase

Horseradish peroxidase

Cytochrome P-450cam

P-420PH 3PH 6Cytochrome c oxidase

Cytochrome c peroxidaseBovine hear tpH 6.4pH 8.5In the absence of substrates, inhibitors or effectors.Two bands were fused; see text.A small peak.

cm"1953 (15), 1933 (15) This paper1965 and 1958 (25)b1961 (>20)

This paper1951(8) (4, 599)1944 (12), 1931 (11,1)1947.5 (6)1957 (> l o )1938 (l l) , 1925 (10)1938 (- ll) , 1906 (19)1933 (16), 1929 (shoulder)1933 (12), 1905 (17)1963 (11-12), 1942 (19-21Y1940 (13)1966 (-23)1942 (-30)

(22)

(23)

(24)

1963.5 (4)1922 (12.5)1948 (33)


6/7

IR S pec t ra of CO Complexes of Heme-containingioxygenases 1405indoleamine 2,3-dioxygenase. We recently obtained evidenceth at th e enzyme has also a regulatory binding site for L-tryptophan besides the catalytic binding site; additions ofcertain indole derivatives such as skatole and indole stimu-lated the catalytic activity of th e enzyme, when a low concen-tration of D-tryptophan was used as the ubstrate. The detailswill be described el~ ew he re .~rom these findings and resultsherein described, we suggest that the prominent changes inthe infrared spectrum of indoleamine 2,3-dioxygenase is alsocaused by the substratebinding to thecatalytic site.Question then arises as to the mechanism by which boundsubstrate at the catalytic siteaffects the CO stretch bands. I nthis regard, a possible correlation between th e steric structureof CO-heme adduct an d CO stretch ing frequency is notewor-thy. It has been shown by using model systems that Fe- C-0bond is essentially linear in the absence of steric crowding(27,28 ) while th e same bond in hemoproteins is bent or tilteddue to th e steric crowding around t he bound CO (29, 30).When CO stretching frequencies were measured, syntheticheme models such as diethylprotoporphyrinatoiron-l-meth-ylimidazole an d picket fence iron porphyrin-l-methylimida-zole, where Fe -C -0 bonds a re presumably linear, showed thestretch band s aroun d 1970 cm" (12), while th e values forordinary hemoproteins were always lower than 1970 cm"(Table 111).Such lower CO stretching frequencies in hemo-proteins have been ascribed to th e*-back donation from irond-orb ital to CO **-orbital (31-33), of which exte nt isdepend-ent on the steric structure f Fe- C-0 (34). Then , the bservedshifts in the CO stretching frequencies of the dioxygenasesupon su bstrate binding can also be correlated with th e changesin steric structu re of the CO -heme adduct caused by thesubstr ate binding at th e atalytic site. In these dioxygenases,the catalytic binding site for L-tryptophan is considered toexist in a close proximity to th e heme iron; the bound L -tryptoph an and oxygen at the heme must react with eachother to yield the reaction product. It is not surprising there-fore that configuration of the CO-heme adduct is altered bythe steric pressure of the bound su bstrate itself or by tha t ofamino acid residue(s) of th e protein which is forced to moveby the substratebinding to thecatalytic site.Th e observed insensitivity of the CO stretch bands againstpH is worthy of note. Since hydrogen bonding is known to besensitive to pH, he finding may suggest tha t changes inhydrogen bond network around CO -heme adduct do not playa crucial role in he changes of CO stretching frequency.Effects of other electronic factors such as electron densitydistribution in the iron-porphyrin may not be so large asobserved in this study, because our recent experiments onPseudomonas L-tr yptop han 2,3-dioxygenase showed elativelysmall shifts of the CO stretching frequencies upon substitu-tion of the heme-side chains with various electron withdraw-ing or donatinggroups? Influence of the proximal ligand mayalso be small, since CO com plexes of cytochro me P-450 whichhas a hiolate anion at th e proximal position shows theirstretch band s in th e same region to those of other hemopro-teins with an imidazole nitrogen at the site (24, 25) (Table111). On the basis of theseand foregoing discussions , wepropose that the bserved changes in CO stretching frequencyis caused by the changes in the steric crowding around theheme associated with the binding of a substrate to the cata-lytic site. Our recent studieson magnetic and naturalcirculardichroism of these enzymes support th is view (6, 7). On theother hand, the smaller influence of L-tryptophan at heregulatory site on the infrared spectrum s probably due to itsindirect and weak action to th e CO complex through theconformational changes in theprotein.

Most of our present results are well explained in terms ofabove postulations. However, we could not explain why theaddition of D-tryptophan had no significant effect on th e COstretch band s of indoleamine 2,3-dioxygenase. Similar inert-ness of D-tryptophan on the CO complex has also been noticedin our recent studies on magnetic and natura lcircular dichro-ism spectra of this enzyme (7). These findings were in contrastwith those of L-tryptop han which always givesmarked effecton th e spectrum irrespective of th e valence, spin, an d ligandbinding states of the enzyme. Being a substrate,D-tryptophanshould bind to the catalytic site during th e catalysis to forma ternary complex of 02 and D-tryptophan with the enzyme,as has been observed with L-tryptophan as substrate (35,36).Th en , he formation of a ernary complex ofCO and D-tryptophan with the enzyme can also be expected. Neverthe-less, we could not obtain any evidence for th e binding of D-tryptophan with the CO complex of indoleamine 2,3-dioxy-genase as stated above. In this regard, the following possibil-ities may be considered (a) D-tryptophan can bind to th ecatalytic site but does not affect the configuration of F e- C -0nor cause the changes in steric crowding around the hemebecause of its D -configura tion, and ( b ) D-tryptophan is prac-tically unable to b ind w ith th e CO complex because of its lowaffinity toward t he complex. In this connection, a differencebetween the structures of CO and O2 adducts of heme ironhas been noted. It has been shown tha t Fe -0- 0 bond isinherently ent even without steric hindrance (37, 38),whereas tha t of Fe- C- 0 is essentially linear in th e absence ofsteric pressure (27, 28). Experiments to discriminate thesepossibilities are now under progress.Finally, detection of a t least two CO stretch band s forPseudomonas enzyme without L-tryptophan a nd also for ratliver enzyme with a n excess amount of L-tryptophan m ightsuggest the nonequivalency of the hemes in these enzymesunder the experimental conditions. Both enzymes have twoprotohemes/mole of enzymes which are tetramers. Indoleam-ine 2,3-dioxygenase, which is a m onom er with a sing le heme,also showed two bands in th e absence of L-tryptophan indi-cating certain kindsf heterogeneity in its eme-CO structureunder the conditions. Possible effects of such nonequivalencyor heterogeneity on the other physicochemical properties,catalytic and ligand binding activities of these enzymes re-main t o be elucidated.

Acknowledgments-We wish to thank Dr. K. Kuratsuka and hisassociates, National Institute of Health, Japan, for supplying us freshrabbit intestine. W e are also indebted to Y. Tanizaki and A. Sa sakifor their excellent technical assistance.REFERENCES

1. Hayaishi, 0. 1980) in Biochemical and Clinical Aspects of Tryp-tophan Metabolism (Hayaishi, O ., Ishimura, Y., and K ido, R.,eds) pp. 15-30, E lsevier/North-Holland Biom edical Press, Am-sterdam2. Ishimura, Y., Makino, R., and Iizuka, T. 1980) Adu.EnzymeRegul. 18,291-3023. Feigelson, P., and Brady, F. 0. 1974) in Molecular Mechanismof Oxygen Actiuation (Hayaishi, O . , ed) pp. 87-133, AcademicPress, New York4. Maxw ell, J. C., and Caughey, W. S. (1978) Methods Enzymol.64,

5. Caughey, W .S., Houtchens, R. A., Lanir, A., Maxwell, J. C., andCharache, S. (1978) in Biochemical and Clinical Aspects ofHemoglobin Abnormalities (Caughey, W . S., ed) pp. 29-56,Academic Press, New York6. Uchida, K., Shimizu, T., Makino, R., Sak aguchi, K., Iizuka, T.,Ishimura,Y. , Nozawa,T.,and Hatano, M. (1983) J . Biol. Chern.7. Uchida, K., Shimizu, T., Makino, R., Sakaguchi, K., Iizuka, T.,

302-323

258, 2519-2525


7/7

1406 IR Spectra of CO Complexes of Heme-containing Diox yge me sIshimura, Y., Nozawa, T., andHatano, M. (1983)J. Biol. Chem. 27 . Peng, S. -M., and Ibers, J. A. (1976) J. Am. Chem.SOC. 8 ,8 0 3 2 -

8. Makino, R., Sakaguchi, K., Iizuka, T., and Ishimura, Y. (1980)J. 28 . Hoard, J. L. (1975) Porphyrins andhfetalloporphyrins (Smith, K.Biol. Chem. 26 5, 11883-11891 M., ed) pp. 317-380, Elsevier/North-Holland Biomedical Press,9. Alben, J. O .,and Caughey, W. S. (1968) Biochemistry 7,175-183 Amsterdam10. Fager, L. Y., nd Alben,J. 0.1972) Biochemistry 11,4786-4792 29 . Norvell,J. C., Nunes, A. C., and Schoenborn, B. P. (1975) Science11 . Caughey, W. S. (1970) Annu. N . Y . Acad. Sci. 1 7 4 ,1 4 8 -1 5 3 (Wash.D. . ) 1 9 0 ,5 6 8 -5 7 012 . Collman, J. P., Brauman, J. I., Halbert, T. R., and Suslick, K. S. 30 . Heidner, E. J. , Ladner, R. C., and Perutz, M. F. (1976) J . Mol.13. Caughey, W. S., Shimada, H., Choc, M. G., and Tucker, M. P. 31. Caughey, W. S., Eberspaecher, H., Fuchsman, W. H.,McCoy,S,14 . Shimada, H. , and Caughey, W. S. (1982) J. Biol. Chem. 257, 32 . Caughey, W. S.,Barlow, C. H., OKeeffe,D. H., and OToole, M.15 . Iizuka, T., Makino, R., Ishimura, Y., and Yonetani, T. (1985) J . 33. Kitagawa, T., Kyogoku, Y., Iizuka, T., and Saito, M. (1976) J.Biol. Chem. 260, 1407-1412 A m . Chem. SOC.98, 5169-517316. Yamamoto, S.,and Hayaishi, 0.1967)J. Biol. Chem. 2 4 2 ,5 2 6 0 - 34 . Walsh, A., and Loew, G. H. (1982) J . Am. Chem.SOC. 0 4 ,2 3 4 6 -5266 235117. Ishimura, Y., Nozaki, M., Hayaishi, O., Tamura, M., and Yama- 35 . Hirata, F., Ohnishi, T., and Hayaishi, 0. 1977) J. Biol. Chem.zaki, I. (1967) J . Bwl. Chem. 242,2574-2576 252,4637-464218. Makino, R., Sakaguchi, K., Iizuka, T., and Ishimura, Y. 1980) in 36. Taniguchi, T., Sono, M., Hirata, F., Hayaishi, O ., Tamura, M.,Biochemical and Medical Aspects of Tryptophan Metabolism Hayashi, K., Iizuka, T., and Ishimura, Y . 1979)J. Biol. Chem.(Hayaishi, O., Ishimura, Y., and Kido, R., eds) pp. 179-187, 234,3288-3294Elsevier/North-Holland Biomedical Press, Amsterdam 37 . Collman, J. P., Gagne, R. R., Reed, C. A., Halbert, T. R., Lang,19. Feigelson,P., and Maeno, H. (1967) Biochem.Biophys.Res. G., and Robinson, W. T. (1975) J . Am . Chem. SOC. 7, 1427-Commun. 28,289-293 143920 . Schimke, R. T. (1970) Methods Enzymol. 17 A , 421-428 38 . Jameson, G.B., Molinaro, F. S., Ibers, J. A., Collman, J. P.,21. Maxwell, J. C., Volpe, J. A., Barlow, C. H., and Caughey, W. S. Brauman, J. I., Rose, E., and Suslick, K.S. (1980) J. Am. Chem.22. Fuchsman, W . H., and Appleby, C. A. (1979) Biochemistry 18, 39 . Watanabe, Y., Fujiwara, M., Yoshida, R., and Hayaishi,0.1980)23 . Barlow, C. H., Ohlsson, P.-I., nd Paul, K.-G. (1976) Biochemistry 40 . Young, L. J., and Caughey, W. S. (1981) in Oxygen and Oxy-24 . OKeefe,D. H., Ebel, R. E., Peterson, J. A., Maxwell, J. C., and Powers, E. L., eds) pp. 787-790, Academic Press, New YorkCaughey, W. S. (1978) Biochemistry 17,5845-5852 41 . Caughey, W. S., Shimada, H., Tucker, M. P. , Kawanishi, S.,25 . Shimada, H., Iizuka, T., Ueno, R., and Ishimura, Y. (1979) FEBS Yoshikawa, S., and Young, L. J. (1982) in Oxygenase and26 . Yoshikawa, S., and Caughey, W. S. (1982) J. Biol. Chem. 2 5 7 , Coon, M. J. , Ernster, R., and Estabrook, R. W., eds) pp. 429-

258,2526-2533 8036

(1976) Proc. Natl. Acad. Sci. U. S. A . 7 3 , 3333-3337 Biol. 1 0 4 ,7 0 7 -7 2 2(1981) Proc. Natl. Acad. Sci. U. S. A . 7 8 , 2903-2907 and Alben, J. 0.1969) Annu. N. . Acad. Sci. 153,722-73711893-11900 C. (1973) Annu. N. Y . Acad. Sci. 2 0 6 ,2 9 6 -3 0 8

(1974) Biochem.iophys. Res. Commun. 68, 66-171 SOC.102,3224-32371309-1321 Biochem. J . 189,393-40515,2225-2229 Radicals inhemistry and Biology (Rodgers, M.. J. , and

Lett. 98,290-294 Oxygen Metabolism (Nozaki, M., Yamamoto, S., Ishimura, Y.,4 12-420 441, Academic Press, New York

Documents

J. Biol. Chem.-1985-Uchida-1400-6