Oct. 20, 1956
SPECTRAL CHARACTERISTICS OF THE SEMIQUINONE FORMS OF FLAVINS [CONTRIBUTION FROM
THE INSTITUTE FOR
5323
ENZYME RESEARCH,UNIVERSITY O F mrISCONSIN]
Spectral Characteristics of Flavins at the Semiquinoid Oxidation Level' BY HELMUT BEINERT RECEIVED MAY14, 1956 Spectra of flavins were studied over the wave length range 230 t o 1300 mp. Families of curves were recorded during progressive oxidation of the reduced t o the oxidized forms at various conditions of PH and concentration. Absorption bands were observed which are maximally developed at about 50% oxidation. These bands were assigned t o semiquinoid intermediates in their monomeric and dimeric forms on the basis of concentration and temperature dependence. The monomeric semiquinone between $H 2 and 7 is characterized by a band with A,,= 565 mp and below pH 2 by a shift of the base 445 mp for flavin mononucleotide a t pH 6) toward longer wave lengths. The principal abof the main flavin peak (A, sorption of the dimeric forms occurs between 700 and 1100 mp.
On the basis of potentiometric titrations, magnetic measurements and spectral observations, several investigators2-8 concluded, 20 years ago, that riboflavin and related compounds are oxidized and reduced in two distinct one-electron steps and therefore pass through a true semiquinoid state of oxidation. The semiquinoid intermediates were found to have a considerable lifetime under certain conditions. Recent studies on a group of enzymes which contain flavin as prosthetic group has provided strong evidence that stable semiquinoid intermediates of these prosthetic flavins may play a significant role in flavoprotein c a t a l y ~ i s . ~As this evidence rests mainly on spectral observations i t was desirable to obtain more information of the spectral properties of the serniquinoid intermediates of simple flavin compounds. This communication contains a qualitative survey of the spectral characteristics of flavins a t different levels of oxidation and under varying conditions of pH, concentration and temperature. The work on the flavoproteins will be described in a separate communication.
Experimental Materials.-Riboflavin was a product of Merck and Co. (U.S.P.), F M P O (sodium salt) of Hoffmann-LaRoche, Inc., and F4D10 of the Sigma Chemical Co. Both F M N and FAD showed only insignificant spots characteristic of other flavins, when chromatographed in two solvent systems." A molar extinction coefficient of 12.2 X 106 cm.2 X mole-' was assigned t o F M N and of 11.3 X 106 X mole-' to F A D at 450 m,u.12,1a On the basis of these coefficients the samples of F M N and FAD used in these studies were estimated to contain 88 and 75%, respectively, of the pure nucleotides (free acid form). The dithionite used was of commercial grade (Mallinckrodt). Apparatus.-For the initial exploratory work the rapid scanning spectrophotometer of the American Optical Companyi4 was u s r t l . Because this instrument played a more ______ (1) This work was supported by a grant (No. NSF-G1772) from t h e National Science Foiindation (2) R Kuhn and T. Wagner-Jnuregg, nu.,67, 361 (1934). (3) R. Kuhn and R. StrBhele, ibid., 7 0 , 7.53 (1937). (1) L . Michaelis. hI. P. Schubert and C . V . Smythe, J . Bicl. C k e m , 116, BR7 (1936). ( 5 ) L.Michaelis and G. Schwarzenhach, ibid., 123, 527 (1938). (6) I,. Michaelis, "The Enzymes," Vol. 11, P a r t 1, J . B. Sumner and K. Myrback, ed., Academic Press, Inc., New York, h-.Y . . 1951, p. 1. (7) K G. Stern, Biochcm. J . , 28, 949 (1934). (5) F. J. Stare, J . B i d . C h e m . , 112, 223 (1935). (9) H. Beinert, Biochim. B i o p h y s . Acta, 20, 588 (1950). (10) Abbreviations used: F h l N , flavin mononucleotide; F A D , flavinadenine dinucleotide. (11) F. L. Crane, S. Mii, J. G. Hauge, D. E. Green and H. Beinert. J . Biol. C h e m . , 218, 701 (1956). (12) L. G . Whitby, Biochem. J . , 54, 437 (1953). (13) 0. Warburg a n d W . Christian, Biochcm. Z.,298, 150 ( 1 9 W . (14) American Optical Company, Instrument Division, Buffalo, New York.
decisive role in the study of the flavoproteins, its specific use will be described in a later communication. IJnder the conditions of the present experiments the lifetime of the intermediates was sufficiently long so that the spectra could be recorded with a Beckman automatic recording spectrophotometer, Model DK 1. This instrument was equipped for linear recording of absorbance and for temperature control of the cell compartment. The wave length scale was not linear. Assignment of wave lengths to maxima and minima was made according to Lewin and Fairbanks.'S Occasional control measurements with a spectrophotometer, Model DU, were made when precise quantitative information was desired. Silica cells of varying light path from 0.5 t o 10 cm. were used and quartz inserts for shorter light paths. The p H was routinely measured after each experiment with a Beckman p H meter Model G. Procedure.-Since in a cuvette with a 1 cm. light path the concentration of total flavin has t o be about 10-8 M for the characteristic bands of the intermediate states t o be recognized, the use of riboflavin for routine work is ruled out because of its low solubility, except at alkaline pH. Most of the spectral studies were carried out therefore with F M N and FAD; 0.016 M stock solutions of these substances were kept frozen and protected from light. Aliquots of these solutions were diluted with appropriate buffers. For the study of the semiquinone band in the range above 500 nip a concentration of flavin and a corresponding light path were chosen such that the product c X di6 was about 2 X 10-3 mole X cm. per liter. At this concentration of flavin the principal absorption band a t -150 mp appears only as a steep end absorption at the left side of the records a t about 600 mp. In the spectral range below 500 mp, flavin concentration and light path were chosen such that c X d was 2 to 3 X 10-6 mole X cm. per liter. The absorbance of the sample against an identical buffer solution devoid of flavin was determined at the starting wave length in a spectrophotometer (Beckman Model DU) and the recording instrument was sct accordingly. Samples in the cylindrical cuvettes of 5 and 10 cm. path length were gassed with helium while the snialler cuvettes mere filled with helium above the liquid level and capped with a rubber or paraffin cap un!ess the quartz inserts were used. The spectrum of the oxidized forni was then recorded. Thereafter, 10 t o 20 pl. of a dithionite solution were added under gassing t o reduce the flavin completely. The dithionite solution was prepared as follows: 0.5 g. of the anhydrous salt was dissolved in 4.5 ml. of a buffer solution 2 t o 4 times more concentrated than that used in the experiment and 0.5 ml. of 6 N KOH was then added. The solution \vas cleared by centrifugation and kept well stoppered in ice. Such a solution is usable for at least one day and does not change the pH of the samples noticeably when adticd in the specified qnantities. After dithionite addition the spectrum of the reduced form was recorded. Thereafter the sample was shaken gently and a few air bubbles were admitted when necessary by slightly lifting the cap. The characteristic color of the intermediate (see Table I ) in most cases then became visible t o the eye. Spectra were recorded in succession with intermittent agitation of the sample until the flavin was completely oxidized. Unless the samples were agitated there was rarely a significant change in absorption during n single scan of 2 to 3 minutes duration. This could he shown b y repeated scanning without intermittent agitation. (1.5) S. Z. Lewin and R. H. Fairbanks, Atzal. Chem., 22, 2020 (1955). (1G) c, concentration nf flavin in moles per liter: d . light path in cm.
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HELMUT BEINERT TABLE I ABSORPTIONMAXIMA,MINIMA AND ISOSBESTIC 9H Medium
0 1 M HC1
2.4 0.085 M phosphate
6.1 0.25 .VI citrate
VOl. 78
POINTS O F
FMN
8.9 0.10 M histidine
12.0 0.125 M phosphate
Oxidized form Amax 267,385 267,370,448 267,375,445 267,375,447 269,353,450 240,303,398 241,303,400 241,303,400 241,295,397 Oxidized form A m i n 238,303 248 251 256 256 Reduced form Amsx 250," 317" ?" 233,255,289,330 234,255,289,330 234,262,289,330 233,264,282,320 Isosbestic points Semiquinoid form monomer Xmsx 503b 525b 565 ............. ............. dimer Xmar 830 (FAD850) 840 (FAD 850) 880 (FAD 900) 1010 (FAD 1040) 770 (FAD 800) visible color" Brownish red Greenish brown Brownish green Greenish yellow Slightly greenish yellow a The spectrum of the 100% reduced form was not recorded, the values are estimated. The original spectrum of the oxidized form was different from that of the sample after final reoxidation (see Fig. 6); sharp isosbestic points were therefore not obtained. Maxima of difference spectra: semiquinone form minus oxidized form. The visible color is composed of contributions from the three components in equilibrium, R, S and T (cf. Scheme 1) and can therefore not be considered as the color of the semiquinone proper. At pH levels above 8 reduced flavins are reoxidized readily and very gentle shaking and fast experimentation are therefore necessary. At pH levels below 6 the intermediate stages are quite stable. A t PH levels below about 3 the basic procedure which has been described above fails because turbidity develops soon after addition of dithionite. This difficulty can be partly circumvented by reducing the flavin only partially so that no excess dithionite remains, by working rapidly and by measuring the spectrum at various phases of oxidation or reduction successively with several equal aliquots of the same original sample. As an alternative one may use reducing agents other than dithionite. Sodium amalgam (0.570) was used successfully for this purpose as was also metallic zinc (below p H 1). With the amalgam, Dowex 50 ( H + ) was added in small portions to control the PH. Strong buffers were used and the pH was frequently checked with strips of a narrow range indicator paper.'? -4ccording to measurements with the glass electrode, the pH could thus be maintained within 0.2 unit of the pK of the buffering ion. However it was very difficult t o reduce the flavin completely, clarify the solution and transfer i t to a cuvette without partial reoxidation of the flavin. There was also a small unexplained loss of flavin tinder these conditions. Complete reduction with Zn is likewise difficult. Z n + + ions may be removed with Dowex 50. This necessitates, however, clarification and transfer. I t was therefore preferred in the present work to reduce the flavin in the cuvette by a piece of Zn suspended from a rubher band, remove t.he metal, leave Z n + + ions in the solution arid thus avoid a transfer. When the spectrum below 400 mp had to be recorded, special precautions had to be taken in the use of dithionite. Dithionite has a strong absorption band with Amax 313 m.u18 at alkaline pH which is shifted to 316 mp a t PH 8.1. The molar extinction coefficient was found to be close to 6 X 106 X mole-' in agreement with Hellstrom.'* This hantl will disappear completely on aeration and only a slight absorption will remain which rises exponentially toward shorter wave lengths. The residual absorption of a O.l", solution of the dithionite used a t a 1 cm. light path and pH 6.1 was 0.30,0.11,0.05,0.02 and
