FOR AN INTERMEDIATE IN THE OXIDATION-REDUCTION OF FLAVOPROTEINS

Previous publications (14) have drawn attention to the fact that a broad absorption band emerges between 500 and 650 rnp when one of the acyl dehydrogenasesl is reduced by substrate. It was found more recently (6) that a band of the same int.ensity and charact.erist.ics appears during t,he few seconds in which reduction of t.hese flavoproteins by dit,hionite proceeds or during subsequent oxidation by oxygen. The same phenomenon could be observed with the free flavin compounds FAD2 and FMN (7). Evidence adduced in a study of the spectra of FAD and FMN during oxidation-reduction (7) and also work by previous investigators (8-13) suggest that the new absorption band may be ascribed to a semiquinoid form of flavin. This paper describes the techniques which were used for recording rapid spectral changes and t,he spectra obtained during oxidation-reduction of three flavoproteins. In addition to one of the acyl dehydrogenases, n-amino acid oxidase of snake venom and t.he old yellow enzyme of yeast were investigated. The old yellow enzyme is of particular interest, as Haas (14) has described a red intermediate of this enzyme which appeared upon partial reduction and was thought to be a semiquinone.

Previous publications (14) have drawn attention to the fact that a broad absorption band emerges between 500 and 650 rnp when one of the acyl dehydrogenasesl is reduced by substrate.
It was found more recently (6) that a band of the same int.ensity and charact.erist.ics appears during t,he few seconds in which reduction of t.hese flavoproteins by dit,hionite proceeds or during subsequent oxidation by oxygen.
The same phenomenon could be observed with the free flavin compounds FAD2 and FMN (7). Evidence adduced in a study of the spectra of FAD and FMN during oxidation-reduction (7) and also work by previous investigators (8)(9)(10)(11)(12)(13) suggest that the new absorption band may be ascribed to a semiquinoid form of flavin.
This paper describes the techniques which were used for recording rapid spectral changes and t,he spectra obtained during oxidation-reduction of three flavoproteins.
In addition to one of the acyl dehydrogenases, n-amino acid oxidase of snake venom and t.he old yellow enzyme of yeast were investigated.
The old yellow enzyme is of particular interest, as Haas (14) has described a red intermediate of this enzyme which appeared upon partial reduction and was thought to be a semiquinone. EXPERIMENTAL
L-Amino acid oxidase was prepared from the venom of Southern water moccasin.
An initial heat denaturation of impurities was carried out according to Singer and Kearney (16). The protein in the clarified supernatant fluid was precipitated with ammonium sulfate at 90 per cent saturation and subjected to zone electrophoresis on starch in 0.1 M Tris acetate of pH 7.2. According to the electrophoretic mobility given by Singer and Kearney, the enzyme would be expected to move towards the anode under these conditions. However, on the starch column, the enzyme moved in the opposite direction under the conditions used, probably because of a considerable endosmotic flow.
The anode was therefore connected to the top of the column and the cathode to the bottom.
After recovery from the column, the enzyme still contained a considerable amount of contaminating protein, but it had the spectrum shown by Singer and Kearney (17), and the absorption at 465 nm was reduced instantaneously to 36 per cent of its original value on addition of n-leucine.
The preparation was therefore considered satisfactory for the planned spectral observations. The preparation of the old yellow enzyme was kindly made available by Dr. Carl S. Vest&g of the University of Illinois and was of about 85 per cent purity (18).
TPN was a product of t,he Pabst Laboratories and TPNH of the Sigma Chemical Company.
The compounds were listed by the manufacturers as of 90 per cent or higher purity.

Apparatus
The Beckman spectrophotometers model DU, DK-1, and DUR were used with accessories for the control of temperature and for the reduction of sample size according to Lowry and Bessey (19). When immediate readings or recording was desired, additions were made on the end of plastic-coated wires.
These wires had a loop at one end which was oriented in a horizontal plane and fitt,ed into the lumen of the spectrophotometer cell. Addition and mixing could thus be accomplished in about half a second.
For the measurement of rapid spectral changes, the rapid scanning spectrophotometer of t,he American Optical Company was found of great value. This instrument (called henceforth the A. 0. spectrophotometer) scans the spectral region from 400 to 700 nm 60 times a second and plots per cent t,ransmittance on a fluorescent screen by means of a cathode ray beam. The standard cell holder of t,his instrument was replaced by a Beckman cell holder, to which a cooling coil was soldered.
Samples A diaphragm with a circular hole of 1 cm. diameter was fastened at the sample position of the cell holder, which permitted only the bright center of the light beam to pass. A 3 ml. Corex cell of 1 cm. light path was used for the sample.
The cell was lifted up on an aluminum block which was inserted into t,he holder, so that the bottom of the cell was just below the hole of the diaphragm.
The minimal fluid volume needed with this arrangement was 1.2 ml. Further reduction of the sample, and consequently also of the light beam, led to an unacceptable distortion of the 109 per cent transmittance line in the absence of absorbing material.
The transmittance of standard samples was measured at several wave lengths in a model DU and in the A. 0. spectrophotometer. The average deviation found was 2.5 per cent, and the greatest deviation was 4.5 per cent of the absorbance which was determined in the model DU.
Although this performance is satisfactory, the A. 0. spectrophotometer is much less reliable in its wave length setting.
This is shifting continuously. The wave length scale does not shift as a whole, but individual areas expand or contract.
Some of these changes were found t,o be reproducible and to depend upon the time the instrument was in operation in each experiment.
The wave length setting was therefore recorded and adjusted with the aid of a didymium filter before and after every reaction sequence. The tracings which appeared on the screen were recorded on Tri-X film with a 16 mm. movie camera mounted 2 feet away from the screen. The camera lens was adjusted to an f of 1.9. An exposure of sixteen frames per second was found most suitable for the work.
A mask was placed in front of the screen to cover bright spots outside the coordinate field, and the room light was subdued.
It was not necessary to shut the sample compartment.
Additions were made from the outside into the previously placed cell with a specially bent, plastic-coated wire.
The limitations of this technique for the recording of rapid spectral changes are (a) the time required for mixing and (5) the memory of the screen which is of the order of half a second.

Methods
The analytical methods and the coefficients used for the determination of protein and flavin were those described in a previous publication (2) or in the subsequent paper (29).
bleached by dit,hionite and 53 per cent by octanoyl CoA at lo-+ M concentration.
The enzyme was first reduced with 5 11. of a 4 per cent dithionite solution.
The photographic records of some of the successive oxidation stages were superimposed and ret,raced in Fig. 1 of mixing, full development at 16 to 22 seconds (Curve 5), and reverted again (Curves 6 and 7) close t.o the original level within 2 minutes (Curve 8). On shaking with air, t,he spectrum passed once more through the same successive stages in reverse order. After reoxidation of the enzyme the spect,ral changes, which occurred on addition of substrate ( Fig. 2), were recorded. 0.2 pmole of octanoyl CoA was added. The band appeared again at about the same initial velocity as upon addition of dithionite. 75 per cent of the total band height, which developed after addition of octanoyl CoA, was reached within 2 seconds.
A further increase occurred within 1 minute (Curve 2). The enyzme solution had changed from a bright yellow to a brownish green color. After 1 minute 10 ~1. of a 4 per cent dithionite solution were added which led immediately to a new equilibrium (Curve 3). The absorption at 447 rnF dropped and the band at 565 rnp increased slightly.
Thereafter, reduction of the flavin proceeded slowly in the course of about 30 minutes to the stage of Curve 4. Then 0.4 pmole of crotonyl CoA was added, which led within 2 seconds to a reversal (Curve 5) and after 2 minutes to Curve 6. Similar records were obtained with the green butyryl dehydrogenase (21,2) and palmityl dehydrogenase (4).
Interpretation of Spectra-The spectral pattern observed when Y is reduced by substrate or reoxidized with the oxidized form of the substrate is strikingly similar to the pattern found when Y is reduced by dithionite and reoxidized with air. A study of free flavins (7) revealed that the spectral characteristics of these compounds during oxidation-reduction at neutral pH were almost identical with those observed with the acyl dehydrogenases. In a previous publication (6), a difference spectrum is shown of the brownish green intermediate which occurs during reoxidation of reduced FAD by air and the oxidized form of FAD. This spectrum is al-most superimposable on a difference spectrum of Y reduced by octanoyl CoA and Y in its oxidized form.
Common to both spectra is a relatively broad absorption band between 500 and 650 rnp with a peak at 560 to 570 rnp. In the spectrum of the free flavin there is an additional broad band in the near infrared region which is not found with the enzymes.
It is remarkable that the band with X,,, 565 rnp has about 20 times t.he intensity with enzyme-bound FAD as with free FAD.
The conclusion was drawn from previous work (7-13) that the band with x n,sx 565 rnp, which occurs during reduction and reoxidation of free flavins, may be ascribed to a semiquinone form of the flavin and the band in the near infrared region to a dimer of the semiquinone.
It may therefore be suggested that the intermediate which occurs upon reduction of the acyl dehydrogenases with dithionite or with substrate and which exhibits properties so strikingly similar to those of the intermediate observed with free flavins is also a semiquinone form of the enzyme-bound flavin.
The characteristic band of free FAD and FMN with h,,, 565 rnp showed no concentration dependence; neither did the 565 rnp band of Y. A solution of Y in 0.03 M phosphate buffer of pH 7.4, containing 1.4 X lo-* M enzyme-bound flavin, was partly reduced with 8.6 X 1O-s M octanoyl CoA. When this solution was diluted ten times with buffer, the intensity of the band at 565 rnp was exactly 0.1 times that observed before.
The strong affinit.y of octanoyl CoA for Y is apparent from this experiment.
As shown previously (6) no indicat.ion of a band in the near infrared region, corresponding to the dimer band of free flavins (7), was obtained with Y. Careful scanning of the near infrared region when a 1.4 X lo-* M solution of Y was reduced by substrate or by dithionite confirmed the earlier result.
It was shown (7) that dimerization of the semiquinone of free flavin is a relatively slow process, and one would therefore not expect enzyme-bound flavin to dimerize readily.
Rate of Formation of Intermediate versus Rate of Over-All Reaction--If t,he assumption is made that the intermediate indicated by the broad band (X,,, 565 mp) does indeed occur as an intermediate in the over-all reaction catalyzed by the acyl dehydrogenases and t,he electron-transferring flavoprotein, then it has to be shown that the rate of formation of the intermediate is at least as rapid as the rate of the over-all process.
That this is indeed the case was shownin an experiment with Y'.
A preparation of Y' which had been subjected twice to zone electrophoresis on starch, and which had a riboflavin content of 0.52 per cent, was used. This preparation had been stored in the frozen state for approximately 1 year and had a specific activity of 24 when assayed under standard condit,ions (2) at 30" with octanoyl CoA as substrate and in the presence of an excess of ETF and indophenol.
At 8' a specific activity of 3 was found. From the flavin content the turnover number was calculated to be 7 moles of substrate oxidized per mole of flavin per minute at 8". 1.3 mg. of the same preparation were dissolved in 0.25 ml. of 0.04 M phosphate of pH 7.0 and placed in the cell compartment of a recording spectrophotometer, Beckman model DUR, which was maintained at 8". The chart speed of the recorder was set at 8 inches per minute.
To the enzyme preparation 0.012 pmole of octanoyl CoA was added on the tip of a st,irring wire.
The final concentration of octanoyl CoA was the same as that used in the assay in which the specific activity had been determined.
The band at 565 rnp appeared immediately and reached 96 per cent of its full development in 1.5 seconds. Since the enzyme was found to turn over once in 60/7 = 8.6 seconds at this temperature, the velocity of format,ion of the intermediat,e is more than sufficient to account for its participation in the over-all reaction catalyzed by the enzyme.
A more thorough discussion of the possible mode of participation of this intermediate in the over-all reaction and a more detailed interpretation of the spectral changes recorded in Fig. 2 will be attempted in a subsequent paper (20), which contains additional information essential to such a discussion.
Conditions for Formation and Disappearance of Intermediate-The characteristics of the appearance of the band between 500 and 650 rnl.c on addit,ion of substrate are the same as those of the disappearance of t,he principal flavin band at about 450 rnp which will be more thoroughly discussed by Beinert and Page (20). The band can also be produced when the flavoproteins are first reduced by an excess of dit,hionit,e and when the reaction product, an a,p-unsaturated fatty acyl CoA, is then added. When the band is produced in this fashion, there is always a marked increase in absorption at about 450 mp. For instance, the addition of A2s3-octenoyl CoA will raise the absorption of dithionite-reduced Y at 447 rnp to a level similar to that reached when Y is reduced by octanoyl CoA (3).
When t,he band has been produced in the presence of the oxidized or reduced form of the substrate, it will only disappear concomitant with a pronounced change of the oxidat,ion state of the flavoprotein.
Such a

INTERMEDIATE IN FLAVOPROTEIN CATALYSIS
change may be brought about by oxidat.ion of the dehydrogenases with oxidizing agents, which are able to act in the presence of substrate or by displacement of the substrate and further reduction of the enzyme by dithionite, as discussed in the following paper (20). However, the ad-diGon of enoyl hydrase, /3-hydroxyacyl dehydrogenase,', 3 Mg++ ions, and an excess of DPN is without influence on the band even when the band has been produced with amounts of octanoyl CoA less than stoichiometric with the enzyme-bound flavin. Free A2~3-octenoyl CoA is readily converted to @-ketooctanoyl CoA under these conditions. This shows that the presence of free cr,&unsaturated fatty acyl CoA is not needed to maintain the band, and whatever of this compound has been committed to the production of the band is no longer available to enoyl hydrase.
Agents like p-chloromercuribenzoate and dinitrophenol were without effect on the band. Extinction Coe$cient of Intermediate-The intensity of the absorption band at 500 to 650 rnp is proportional to the extent of bleaching of the prosthetic flavin by substrate.
As will be shown (20), the extent of bleaching by different substrates is inversely related to the K, of the substrate. The intensity of the band or, by the same token, the concentration of the intermediate follows therefore the same inverse relationship to the K, of the substrate.
The spectrum of the pure intermediate has never been obtained since there are always several absorbing species present in equilibrium (7). A minimal value for the molar extinction coefficient can be derived from the experiments reported by Beinert and Page ((20) Table I).
It is shown there that small amounts of octanoyl CoA react stoichiometrically with Y. This reaction leads to a decrease in absorbance at 447 rnp and a parallel increase at 565 rnp. If it is assumed that all the flavin which is bleached by octanoyl CoA is converted to the intermediate in a 1: 1 stoichiometry, a minimal value of the extinction coefficient can be calculated from the ratio of the absorbance changes at the two wave lengths and the known value of the molar extinction coefficient for the difference between oxidized and completely reduced flavin at 447 rnp, i.e. 10.3 X lo6 sq. cm. X mole-1.4 If the lowest observed ratio, 3.1, is used, we obtain 10.3 X lo6 sq. cm. X mole-l divided by 3.1 = 3.3 X lo6 sq. cm. X mole-l as a minimal value for the intermediate. achieved. The original spectrum (Curve 1) was restored as soon as the oxidation was completed.
A slight temporary increase in absorption in the 520 to 650 rnp region was again observed during this oxidation.
Reduction of this enzyme by dithionite was t.oo rapid t,o furnish definite evidence for the typical band in the 520 to 650 rnp region which appeared on reduction with substrate.
Reduction by dithionite is not readily reversible with this enzyme.
Rate of Formation of Intermediate versus Rate of Over-All Reaction-The turnover of the enzyme preparation used was not determined.
An estimate may, however, be made when the figure given by Singer and Kearney is used. According to these authors, 1 mole of enzyme oxidizes 3100 moles of substrate per minute at 38" in the presence of 0.01 M L-leucine (16).
If the temperature coefficient of the reaction is assumed to be of the order of the coefficient found for Y', the turnover at 8" would be about 200 per minute or 3 per second.
Singer and Kearney consider a subst.rate concentration of 3 X 1O-3 M as optimal, "beyond which there is a definite decline" in rate.
In the present work the appearance of the intermediate was follow'ed aerobically at a 7 X 1W M substrate concentration.
L-Amino acid oxidase is, in contrast to Y and Y', very readily autoxidizable in the presence of substrat.e.
In the experiment of Fig. 3, the band in t,he 520 to 650 rnp region developed to 50 per cent of its full height in 2 seconds and to full height in 4 seconds.
In view of the unfavorable substrate concentration in this experiment, the presence of oxygen, and the uncertainty of the temperature coefficient, the observed discrepancy of rates does not appear large enough to rule out the participat,ion of t,he intermediate in the over-all reaction.
Old Yellow Enzyme. Spectral Evidence for Intermedia& mg. of enzyme were dissolved in 1.4 ml. of 0.1 Y phosphate of pH 7.0. The spectrum was recorded wit,h the A. 0. spectrophotometer.
Reduction with dithionite was very rapid, and no indication of an intermediate was obtained. Reduction with TPNH proceeded more slowly within a few seconds, and satisfactory records were obtained. A small rise in absorpbion between 520 and 650 rnp was observed, but could not be considered unambiguous in view of the inherent instability of the A. 0. spectrophotometer and possible interference by turbidity in the concentrated protein solution. It should be pointed out, however, that at the concentration of flavin used in this experiment,, with FMN or FAD likewise no clear indication of the intermediate (X,,, 565 mp) can be obtained (7). There was no indication of an intermediate elsewhere in the spectrum (400 to 700 rnp) which satisfied the criteria discussed in a recent publication (7). Experiments were then conducted at a higher flavin concentration. 0.015 prnoleb of enzyme was dissolved in 0.14 ml. of 0.1 M phosphate of pH 7.4 (Fig. 4, Curve 1). The enzyme was first reduced by dithionite (Curve 2) and reoxidized by air. 0.05 pmole of TPNH was then added (Curve 3). Slow reoxidation of the enzyme took place during the measurements . Curve 3 does therefore not represent the state of maximal reduction by TPNH.
It is shown here only to emphasize that under these conditions no increased absorption at 540 to 650 rnp is observed.
Thereafter 0.1 rmole of TPNH and 0.2 rmole of TPN were added. The gas space in the cuvette was filled with helium, and a rubber cap was inserted . Aft,er a rapid partial reoxidation of the flavin on addition of TPN, a very slow reoxidation took place, which did not interfere with the recording of the spectrum (Curve 4). The absorption at 540 to 650 rnp, however, began to rise as soon as TPN had been added to the reduced enzyme and reached a maximum within about 5 minutes.
As oxidation of t,he flavin proceeded, the absorption at 540 to 650 rnp declined again after about 20 minutes.
The maximal change of absorbance occurred between 560 and 580 rnp and did not exceed a value of 0.025 absorbance unit. carried out under corresponding conditions. In no case was there a distinct red color visible, and the absorption at 465 rnp was always higher than t.hat at 475 rnp.
The small absorbance changes between 540 and 650 rnp which were consistently observed have to be evaluated crit,ically as t.o their significance. In concentrated protein solubions such as used here, t,he development of turbidity may seriously interfere with exact, measurements of small absorbance changes specifically after an addition has been made under stirring. The following facts confirm that the measurements are indeed valid ISTERMEDIATE IN FLAVOPROTEIN CATALYSIS and the values obtained are significant: (a) The experiment, has been repeated four t.imes with the same results.
(6) Turbidity as an int.erfering factor can be ruled out, as the absorbance at 700 rnp actually decreased slightly when the absorbance at 540 to 650 rnM increased (cf. Curves 1 and 4). (c) The increase in absorbance did not occur immediat.ely after addition of TPN under stirring, but slowly thereafter in the course of several minut,es.
(d) The increase in absorbance at 540 t.o 650 rnp, as well as the slight decrease at 700 rnp, was reversed as oxidation of the flavin proceeded.
Although the small absorbance changes observed are considered significant, they could not, without, our awareness of the phenomenon from the work with FAD, FMN, and ot.her flavoproteins, bear much conviction by themselves. The spect.rum of the intermediate recorded by Haa. shows an increase in absorpt.ion between 540 and 600 rnp as it was observed in the present work also, but this increase extends towards shorter wave lengths.

Comparison of Present
The absorpt,ion maximum is thus shifted from 465 to 475 rnp and the intermediate shows t,herefore a red color. Further work will have to provide an explanation for the spectral difference between the intermediate of Haas and that observed in t.he present work. It, appears possible that under t,he experimental conditions of Haas an addit.ional component, contributes to the basic spectrum of t,he semiquinoid intermediate lvhich was exclusively seen in the present work.

DIscussIox
The records presented above show that the int,ermediat,e which is characterized by a broad absorption band between 500 and 650 rnp, and which was first observed with the acyl dehydrogenases, can also be demonstrated with the flavoproteins L-amino acid oxidase and old yellow enzyme. This is not surprising as it has been shown to occur during oxidation-reduction of free flavin.
It may rather be postulated t,hat one should be able to observe the absorpt,ion band wit,h all flavoproteins under suitable conditions. There are dat.a in the literature which are of interest in this context. Dolin (23) has observed an absorpt.ion band with a bact.erial flavoprotein which is strikingly similar to that described here. Mahler and Elowe (24) and Horecker (25) have published spectra for two cytochrome reduetases which show an increased absorption in the spectral region of 500 to 600 rnb on reduction with substrate.
Some quantitative considerations may explain why the band at 500 to 8  We thus obtain the index, -AA~M) m,, (100 per cent reduction)/A&as mp (50 per cent, reduction), for the relative concentration of intermediat,e which may be maximally present in equilibrium with t,he ot,her forms of flavin.
The lower t,he ratio, the higher is the attainable concentration of the intermediat,e. The values for the ratio are 70 for free FAD and FMN, 35 for the old yellow enzyme, 14 for n-amino acid oxidase, and 4 to 5 for Y and Y'.
It is evident from a comparison of these figures that the acyl dehydrogenases were ideally suited for the detection of the phenomenon. Free FAD, for example, would have to be used at a concent.rat,ion corresponding t.o an absorbance of about 8 at 450 rnp to yield a rise of 0.1 absorbance unit at 565 rnM during partial reduction. With the old yellow enzyme an absorbance of 4 at 465 rnp would be required to obtain a rise of 0.1 at 565 ml.c. On the basis of the minimal value for the molar extinction coefficient, of the int.ermediate, 3.3 X 103 sq. cm. X mole-l, which is derived from experiments described subsequently (20), an absorbance increment of 0.1 at, 565 rnp would correspond to a concentration of the intermediate of 3 X lo+ M.
It is obvious t.hat excessive amounts of material may be necessary in attempts t,o recognize the intermediate with some of the flavoproteins. It is also obvious t,hat the acyl dehydrogenases offer the greatest promise for physical studies in which it will be attempted to establish that the intermediate is indeed a semiquinone as suggested.
The concentration, as well as the stability of the intermediate, is extraordinarily high with these enzymes. SUMMARY Spectra have been recorded of several flavoproteins during reduction with substrate or dithionite by a rapid scanning technique.
Records of the yellow acyl dehydrogenase (Cd to C,,)l from pig liver, L-amino acid oxidase from snake venom, and the old yellow enzyme from yeast are presented, which show the appearance of a t.ransient int.ermediate during oxidation-reduction of these enzymes. The intermediate is characterized by a broad absorption band in the region of 500 to 650 rnp, which is typical for the semiquinone form of free flavin at neutral pH (7). Since this by guest on March 24, 2020 http://www.jbc.org/ Downloaded from absorption band has been observed during reduction of the acyl dehydrogenases in t,he absence of substrate, it is concluded that t,he band can be ascribed exclusively t.o an intermediate oxidation state of the prosthetic flavin of the enzymes rather than to an enzyme-substrate complex. The condit.ions of formation and stability of the intermediate have been investigated with the acyl dehydrogenases.
The author is indebted to Dr. D. E. Green for his continued interest in this work and to Dr. Jens G. Hauge for assistance and criticism in the early part of this work.
The technical assistance of hrrs. Mildred Van De Bogart and financial support by the National Science Foundation (grant No. NSF-G1772) are gratefully acknowledged.
Addendum-Since submission of t,his manuscript, it could be shown by electron pnramagnetic resonance absorption that free radicals are indeed formed when substrate is added to the acyl dehydrogenases.
A certain background absorption which was observed with t.he enzymes in the absence of substrate is so far unexplained and requires further study. The author is indebt.ed to Dr. R. H. Sands of Stanford University for his interest and kind collaboration in these experiments. BIBLIOGRAPHY