[35] Isolation, synthesis, and properties of 8-hydroxyflavins

[3 5]

8-HYDROXYFLAVINS

241

Roseoflavin Tetraacetate. C26H3INsO10, mp 279-280 °. Absorption spectrum (CHCI3), hmax(EmM): 260(40.7), 500 nm(40.9). The acetate itself is not fluorescent, though the ordinary preparation shows green fluorescence. [a]D, +260 ° (C = 0.500, CHC13). Proton magnetic resonance (PMR), 6 = 7.87s, 6.80s(6,9-H), 3.13s(8aN-CH3), 2.47s(7-CH3), 2.27(s), 2.13s, 2.07s, 1.80s(CO-CH3) (CDC13; internal standard, tetramethylsilane). It is photosensitive in organic solvent, and degraded to 8-methylamino-8-demethyl-o-riboflavin tetraacetate? 4

Biochemical Properties Roseoflavin has weak antibacterial activity against some gram-positive bacteria, especially Staphylococcus aureus, Sarcina lutea, Bacillus cereus, and Bacillus subtilis.' Its antiriboflavin activity was demonstrated in Lactobacillus casei (ATCC 7469) and in the rat. 4 Berezovskii et al. showed that roseoflavin inhibits the biosynthesis of riboflavin by a represser mechanism) 5 Roseoflavin binds with egg white apoprotein with a lower affinity than riboflavin. '6 It is phosphorylated by flavokinase. '7 Though it binds with apoenzymes, it is inactive as coenzyme with lactate oxygenase from Myobacteriurn phlei, TM old yellow enzyme (FMN form), '7 and bacterial lucife,rase (FMNH2 form).'9 14 K. Matsui and S. Kasai, unpublished (1976). is V. M. Berezovskii, A. I. Stepanov, N. A. Polyakova, L. S. Tulchinskaya, and A. Ya. Kukanova, Bioorg, Khim. 3, 521 (1977). le K. Matsui, K. Yanase, and S. Kasai, to be published. 17 S. Otani, Y. Note, Y. Nishina, and Y. Matsumura, Flavins Flavoproteins, Proc. Int. Syrup., 6th, 1979 (in press). ,8 S. Takemori, unpublished (1976). 1~ T. Watanabe, T. Nakamura, K. Matsui, and S. Kasai, Flavins Flavoproteins, Proc. Int. Symp., 6th, 1979 (in press).

[35] I s o l a t i o n , S y n t h e s i s , a n d P r o p e r t i e s Hydroxyflavins

o f 8-

By SANDRO GHISLA a n d STEPHEN G . M A Y H E W

8-Hydroxy-8-nor-FAD has been isolated from purified preparations of electron-transferring flavoprotein from Megasphaera elsdenii 'a in ' S. Ghisla and S. G. Mayhew, J. Biol. Chem. 248, 6568 (1973). 2 S. Ghisla and S. G. Mayhew, Eur. J. Biochem. 63, 373 (1976).

METHODSIN ENZYMOLOGY,VOL. 66

Copyright© 1980by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181966-3

242

FLAVINS AND DERIVATIVES

[35]

which it has been found to occur in variable amounts together with FAD and 6-hydroxy-FAD.3.4 By comparison with normal flavins, it has unusual light absorption and fluorescent properties, some of which it shares with roseoflavin (8-dimethylamino-8-norriboflavin)? It can be degraded enzymically to 8-hydroxy-FMN and further to 8-hydroxy-riboflavin. The chemical synthesis of 8-hydroxy-FMN is straightforward: 8amino-8-norriboflavin6 is a very convenient starting material that is easily converted to the 8-diazoflavinium salt, 6 and which in turn is an appropriate intermediate for the introduction of a series of functional groups in position 8 of the flavin chromophore by Sandmeier-type reactions (Scheme 1; R = ribityl, alkyl, or H; X = OH-, Br-, CI-, -S-, etc.). 8-Hydroxyriboflavin has been synthesized by this procedureT; the 8chloro-, 8 8-bromo-, 9 and 8-mercapto-derivatives of riboflavin a° can be made just as easily. 8-Hydroxyflavins and 8-hydroxylumichromes are also easily obtained by condensation of appropriately substituted o-aminocresols and violuric acid(s) according to Scheme 2 (R = ribityl, alkyl; when R = H an alloxazine type of chromophore with H at position N(I) is obtained). This synthesis is even more straightforward than that of Scheme 1, and it allows the preparation of various N(3)- and N(10)-substituted 8-hydroxyflavins.2 The method fails, however, when N-substituted o-aminocresols and disubstituted violuric acids are used as starting materials. 2 Several 8-hydroxyflavin model compounds have been synthesized according to the sequence of Scheme 2z; 8-hydroxyriboflavin has also been prepared in this way, but details of the procedure have not yet been reported. 11 Thanks to their optical and chemical properties, 8-hydroxyflavin coenzymes have been successfully used a probes for investigation of the

C. D. Whitfield and S. G. Mayhew, J. Biol. Chem. 249, 2811 (1974). 4 S. G. Mayhew, C. D. Whitfield, S. Ghisla and M. Schuman-J6rns, Eur. J. Biochem. 44, 579 (1974). s S. Otani, S. Kasai and K. Matsui, this Volume, Chapter [34], p. 235; S. Kasai, R. Miura, and K. Matsui, Bull. Chem. Soc. Jpn. 48, 2877 (1975). 6 V. M. Berezovskii, L. S. Tul'chinskaya, and N. A. Polyakova, Zh. Obshch. Khim. 35, 673 (1965). r N. A. Polyakove, L. S. Tul'chinskaya, L. G. Zapeochnaya, and V. M. Berezovskii, Zh. Obshch. Khim. 42, 465 (1972). 8 L. S. Tul'chinskaya, T. A. Zhilina, V. D. Klebanova, N. A. Polyakova, L. M. Solidkina, V. A. Mironov, and V. M. Berezovskii, Zh. Obshch. Khim. 42, 1135 (1972). 9 S. Ghisla, unpublished. 1o E. Moore, V. Massey, and S. Ghisla, unpublished. 11 K. Matsui and S. Kasai, J. Nutr. Sci. Vitaminol. 20, 411 (1974).

[3 5]

8-HYDROXYFLAVINS R I

243 R I

o

o SCHEME I

active sites of flavoenzymeslZ; they m a y p r o v e useful in studies on the reaction m e c h a n i s m s of f l a v o e n z y m e s . I s o l a t i o n of 8 - H y d r o x y - F A D f r o m M e g a s p h a e r a elsdenii 8 - H y d r o x y - F A D can be isolated from extracts of electron-transferring flavoprotein z after purification of the e n z y m e from M . elsdenii according to the p r o c e d u r e s described by Whitfield and M a y h e w / z The content of this ltavin in the e n z y m e varies considerably with the preparation, z however, and recent evidence has strongly suggested that the modified r a v i n is an artifact that is generated in the e n z y m e during its purification. The r a v i n can be more conveniently isolated from a crude extract of the bacterium that has been incubated in air for several days. TM Growth and Extraction of Bacteria

The strict anaerobe, M . elsdenii, strain LC 115 (formerly called P e p TM) is grown in cultures of 40-200 liters with lactate and either yeast extract or corn steep liquor/7"~8 An iron salt is added to the medium with yeast extract if the synthesis of ferredoxin is required, ar but this addition has no detectable effect on the subsequent formation o f 8 - h y d r o x y - F A D . The bacterial cells are harvested by centrifugation, and the cell paste is freeze-dried and then ground to a fine powder. The p o w d e r is suspended in water (10 ml) and the mixture stirred magnetically in a b e a k e r for 1 hr at 37 °, and then for 4 days at 4 °.

tostreptococcus elsdenii

lz S. Ghisla, V. Massey, and S. G. Mayhew, Flavins Flavoproteins, Proc. Int. Symp., 5th, 1975 p. 334 (1976). 13 C. D. Whitfield and S. G. Mayhew, J. Biol. Chem. 249, 2801 (1974). 14 S. G. Mayhew, unpublished; C. D. Whitfield, private communication. 15 S.R. Elsden, B~ E. Volcani, F. M. C. Gilchrist, and D. Lewis, J. Bacteriol. 72, 681 (1956). 16 M. Rogosa, Int. J. Syst. Bacteriol. 21, 187 (1971). 17 S. G. Mayhew and V. Massey, J. Biol. Chem. 244, 794 (1969). is D. J. Walker, Biochem. J. 69, 524 (1958).

244

FLAV1NS A N D D E R I V A T I V E S

R

R I

H ~

H~c"

v

[35]

~

II N'a

OH

O

~.

N\

o

SCHEME 2

Acid Treatment of Bacterial Suspension Trichloroacetic acid (22 ml of a 50% w/v solution) is added to 200 ml of the cell suspension at 4°, and the mixture is centrifuged (15,000 g, 20 min). The precipitate is discarded. Subsequent operations with the yellow supernatant are carried out in dim light. Much of the trichloroacetic acid in the supernatant is removed by five extractions with ether (ether: supernatant, 1:1, v/v). The pH is then adjusted from about pH 3.8 to pH 9 with I M KOH. Residual ether is removed by warming the solution to 40° and bubbling air through it for 30 min. The solution is then cooled to 4° and clarified if necessary by centrifugation (15,000 g, 20 min).

DEAE-Cellulose Chromatography The supernatant is applied to a column of DEAE-cellulose (Whatman DE 32, 20 × 1.5 cm) that is equilibrated with 0.1 M Tris-HC1 buffer, pH 8. The column is then washed with 0.1 M Tris-HCl buffer, pH 8, to remove much yellow and fluorescent material, and to expose a faint orange band in the top half of the column. After about 300 ml of buffer have passed through the column, the eluate should be colorless. The column is then washed with 0.3 M potassium phosphate buffer, pH 5.5. This removes more yellow fluorescent material and causes the separation and slow elution of a band showing blue florescence. When all of the blue fluorescent material has been removed, the column is washed with 0.1 N HCI. The orange material on the column is eluted in a sharp yellow band. The acid eluate is neutralized with solid K2COa, and the resulting orange solution is applied to a second column of DEAE-cellulose (20 × 1.5 cm) equilibrated as described above. The 8-hydroxy-FAD is adsorbed at the top of the column in an orange and fluorescent band. The column is washed with 0.3 M phosphate buffer, pH 5.5, and the orange band is slowly chromatographed down the column. When the band is about 8 cm from the bottom of the column (about 300 ml of buffer), the DEAEcellulose is extruded, the orange resin is cut out and suspended in water, the suspension is poured into a new column, and the column is washed

[35]

8-HYDROXYFLAVINS

245

with 50 ml of water. The absorbed material is then eluted with 0.1 N HCI and the yellow eluate neutralized with K2CO3. The yield of 8-hydroxy-FAD in a typical preparation from 20 g dry bacteria is 50 nmol; this represents a very small fraction of the total flavin in the bacterium (between 1.5 and 2/zmol per gram dry bacterial4,19). The mechanism of formation of 8-hydroxy-FAD in the extracts is not known; oxygen seems to be, required because the hydroxylated flavin cannot be detected in bacterial suspensions that are either treated immediately with acid or incubated under nitrogen instead of in air. TM Preparation of 8-hydroxy-FMN from 8-hydroxy-FAD 8-Hydroxy is prepared by treatment of 8-hydroxy-FAD with phosphodiesterase (Naja naja venom from Sigma Chemical Co., St. Louis Missouri). z The 8-hydroxy-FMN is subsequently purified by thin-layer chromatography, 2 or by binding the flavin to apoflavodoxin, either in solution, or immobilized to Sepharose, 2° The separation with immobilized apoflavodoxin is the preferred method because it is rapid, and it can be used to accumulate large amounts of the modified flavin. Chemical Synthesis of 8-Hydroxyflavins 8-Hydroxy-8-norlumichrome (7-methyl-8-Hydroxyalloxazine ) A mixture of 100 mmol violuric acid and 115 mmol 6-amino-o-cresol (Aldrich) in 150 ml of water that contains 100 mmol sodium borate is adjusted to pH 8.5 and then refluxed for 2.5 hr. The dark brown solution is filtered hot, and the filtrate is acidified to about pH 4 with glacial acetic acid ~md cooled in ice. The precipitate is washed with dilute acetic acid, methanol, and acetone, and dried in vacuo to yield about 9 mmol 8hydroxylumichrome. The compound tan be further purified by dissolving it at pH >9 and reprecipitating at pH 4, or by recrystallization of the disodium salt in 1 N sodium hydroxide. 8-Hydroxy-8-Norlumiflavin (7,10-Dimethyl-8-Hydroxyisoalloxazine ) 2 Trifluoroacetic anhydride (32 g, 150 mmol) is added drop by drop over a period of 10 min to a solution of 6.5 g (50 mmol) of 5-amino-ocresoll (Aldrich) in 30 ml of tetrahydrofuran in a flask fitted with a condenser and stirrer. After 3 hr at ambient temperature, the solvent is evaporated in vacuo. Remaining traces of trifluoroacetic acid and anhy19j. L. Peel, Biochem. J. 69, 403 (1958). 2oS. G. Mayhewand M. J. J. Strating,Eur. J. Biochem. 59, 539 (1975).

246

FLAV|NS AND DERIVATIVES

[35]

dride are removed by azeotropic distillation with benzene. The crystalline residue is then dissolved in 50 ml dry tetrahydrofuran; 2.3 g (0.1 mol) sodium hydride are added in portions, and the mixture is stirred for 15 min. Approximately 10 g (70 mmol) methyl iodide are added, and the mixture is stirred for 4 hr at ambient temperature. The solvent and excess methyl iodide are removed in v a c u o , and the residue is hydrolyzed for 30 min at room temperature with 10 ml 0.1 N sodium hydroxide. For the condensation, 6.2 g (0.1 tool) boric acid and 9.4 g (60 mmol) violuric acid are added to this mixture, the pH is adjusted to 8,5, the solution is diluted with water to a final volume of 50 ml, and it is then refluxed for 2 hr. The hot and dark-colored reaction mixture is acidified to about pH 3 with glacial acetic acid and cooled in ice. The precipitate which forms is collected by filtration and suspended in a minimum volume of cold 10% bicarbonate solution. The mixture is filtered, the filtrate heated to boiling, and then treated with 2 N acetic acid until the beginning of precipitation. After cooling, the precipitate is collected by filtration, washed with dilute acetic acid, and dried. Yield: 1.2 g (9%), mp >.340 °. The compound can be recrystallized by dissolving it at pH >10 and precipitating it from the hot solution with acetic acid. Nuclear magnetic resonance (NMR) (CF3COOH): ~ = 8.32 (6-H), 7.70 (9-H), 4.50 (3-H,10-CH3) and 2.62 ppm (3-H,7-CH3). Infrared (KBr): typical bands at 2790, 1690, and 1650 cm-L 8-Hydroxy-8-Norriboflavin

The preparation of t h i s compound according to the sequence of Scheme 1 has been described by Berezovskii et al., 6 while Matsui and Kasai 11 report its synthesis from N-ribitylcresol and violuric acid (Scheme 2), but give no details of the procedure. Berezovskii et al. 6 obtained a yield of 38% in their procedure, but the extinction coefficients of the products were less than 50% of the correct values. 2 The following procedure is adapted and simplified from that of Berezovskii et al.6; it gives pure material in about 75% yield. Solid sodium nitrite is added in small portions to a well-stirred suspension of 200 mg 8-amino-8-norriboflavin at 0°. The additions are continued during about 15 min and until all of the flavin is dissolved to give a deep orange-red solution. This solution is allowed to stand at 0° for a further 15 min and a minimum of solid urea is then added to destroy any excess nitrite. When the evolution of nitrogen ceases, the solution is poured into 100 ml of boiling glacial acetic acid. The decomposition of the diazonium compound is accompanied by a change of color from redorange to yellow-orange. The solution is boiled for 5 min, the acetic acid is evaporated in v a c u o , the red oily residue is dissolved in 20 ml water,

[35]

8-HYDROXYFLAVINS

247

and the pH of the resulting solution is adjusted to about pH 11 with NaOH. The solution contains mainly 8-acetoxyriboflavin which is completely hydrolyzed to 8-hydroxyriboflavin during 10 min at this pH. The pH is then lowered to about 3.5 with glacial acetic acid, and the turbid solution is allowed to stand overnight. The red precipitate which forms is collected by centrifugation and dried in vacuo. The yield is 150 mg. The compound can be recrystallized from trifluoroacetic acid-water. 8-Mercapto-8-Norriboflavin This molecule is obtained by treatment of the diazonium salt described above (Scheme 1) with excess sodium disulfide. A deep violet solution is formed from which a black precipitate of 8-mercaptoflavin separates when the pH of the solution is adjusted to about pH 3. The compound is best isolated as the yellow disulfide after air oxidation. The disulfide is readily reduced back to the mercapto compound by treatment with mild reducing agents such as dithionite, DTT, or borohydride at a pH above', 6. Properties of 8-Hydroxyflavins in Comparison with Normal Flavins Spectral and Chemical Properties 8-Hydroxyflavins can be readily distinguished from normal flavins by their absorption spectra and by the effects of pH on the spectra. They have a single band of absorption of relatively high intensity in the range 400-500 nm (Fig. 1). The nature of the substituent at position N(10) (isoalloxazine-type chromophore) has only a minor effect on the spectrum; the absence of a substituent at this position or the presence of a substituent at position N(1) (alloxazine-type chromophore) causes a shift to shorter wavelengths, as is also observed with flavins and lumicbromes (Table I). The three ionization processes of 8-hydroxyflavins have been assigned with the help of model compounds 2 (Table I); in particular, deprotonation of the 8-hydroxyl group has been attributed to the pK at about 4.8. This surprisingly low value probably reflects the strong electron deficiency of the ravin nucleus. It is apparent from the data of Fig. 1 and Table I that 8-hydroxyl (or its anionic form) functions as an electrondonating group and causes a bathochromic shift in the visible spectrum. When this group is made to be electron-withdrawing by appropriate substitution, by acetylation or tosylation for example, the spectrum becomes similar to that of normal ravin with bands at about 440, 350, and 265 nm7

248

FLAV1NS AND DERIVATIVES

[35]

474 .252

l !~.

_,--'

pH, 3.0 •

___: 4

P

[ " • 472 ! 473

H. 30

/ ~c'l*

,

Ho qo

!,/.

, 422

i

/1 •

/ : I,"!,t

•

U

•

15" :'3oo

3o2

i \\ . 7. .

3Bo :

.~,,

i ;/

t

*

II

1'.

t

i

I

~.~

•

I

I

;

x'~ kti{ :

{

-1" :J/ . / "

\ .,,. ', ,.

"\\;. \ ...

,

2OO

30O

40O

500

Wovelengih (nm) FIG. 1. Effects of pH on the absorption spectra of 8-hydroxyflavins. The curves show spectra of 8-hydroxylumiflavin at the pH values indicated, except for curve ( ..... ) which is the spectrum of 8-hydroxy-FMN at pH 6.5 in 0.05 M phosphate buffer. Adapted from S. Ghisla and S.G. Mayhew, J. Biol. Chem. 248, 6568 (1973).

In contrast to 6-hydroxyflavins,4'21 but like normal flavins, 8-hydroxyflavins are fuorescent; the wavelength of the emission maximum is in the range 500-530 nm, but it varies with pH. It should be noted that there is a basic difference in the fluorescence behavior of 8-hydroxyflavins and normal flavins. The ionization at position N(3)-H (pK - 10) causes a complete quenching of the fluorescence of normal flavins, but a roughly 2-fold increase in the fluorsecence of 8-hydroxyflavins. This difference can be exploited in analyses for 8-hydroxyflavin. Substitution of 8-CH3 by -hydroxyl also has a marked effect of the electron spin resonance (ESR) spectrum of the flavin radical cation2; the total linewidth of the signal is reduced by 8 G, while the hyperfine structure is also altered. The chemical reactivity of 8-hydroxyflavins parallels in a qualitative way that of normal flavins. 2 The chromophore is reduced by common reducing agents such as dithionite or sodium borhydride. The reduction 21 G. Sch611nhammer and P. Hemmerich, Eur. J. Biochem. 44, 561 (1974).

[35]

8-HYDROXYFLAVINS

249

proceeds through intermediate radicals, as can be observed at low pH, or when the 8-hydroxyflavin is bound to apoenzymes. The chromophore of 8-hdyroxyflavin is photochemically active, with the result that the side chain of the riboflavin derivative can be photochemically removed to form 8-hydroxylumichrome. 1 The redox potential of 8-hydroxylumiflavin is -0.332 V at pH 7 and 25°, x4 and it is therefore about 0.12 V more negative than that of normal lumiflavin (Table II). The plot of redox potential vs. pH for the hydroxylated ravin shows an increase of slope with increasing pH around pH 5.7; the slope then decreases again around pH 6.5. These changes presumably reflect the ionizations of the 8-hydroxygroup and additional ionizations corresponding to the ionizations at pK values of 8.6 and 6.7 in the semiquinone and hydroquinone, respectively, of normal flavins. The different electrophoretic and chromatographic properties of 8hydroxyflavins and normal flavins allow an easy differentiation; the different effects of pH on the electrophoretic mobilities reflect the ionization of the 8-hydroxyl group (Table III).

Properties of Complexes of 8-Hydroxyflavins with Apoflavoproteins 8-Hydroxy-FAD is bound by the apoenzymes of electron-transferring flavoprotein and D-amino acid oxidase; 8-hydroxy-FMN is bound by apoflavodoxins, lactate apooxidase, and apoenzyme of old yellow enzyme. 2,3,12 The shape of the absorption spectra of the complexes, their fluorescence properties, and the effects of pH on the spectroscopic properties of the complexes vary with the apoenzyme, and provide information about the environment in the binding site. Comparison of the shape of the absorption spectrum with those of model compounds can be used to determine whether the proton in the neutral ravin is at C(8)-O or N(1)--C(2)12; the pK of the bound ravin provides an indication of the charge environment in the flavin site. In some cases, the spectroscopic changes that occur during binding are analogous to those observed during binding of the native ravin. For example, when 8-hydroxy-FMN is bound by apoflavodoxin from M. elsdenii at pH 8, the absorbance maximum is shifted to longer wavelengths and decreased in intensity, and the fluorescence is completely quenched. The p K at 4.8 in the free flavin is shifted to 6.1 in the complex, an observation that is in accordance with other observations which suggest that the flavin-binding site is negatively charged. Similar effects are observed during the binding of 8-hydroxyFMN to apoflavodoxins from Desulfovibrio vulgaris and Azobacter vinelandii. In contrast, the spectroscopic properties of the complexes of 8hydroxy-FMN with apo-old yellow enzyme and lactate apooxidase are

Z <

~ ,-,=. ©

o,

m

.4 I.-

8

r~

&

.=

D <

Z <

M

t"q t"q

<

~1 ~

~

~

~

~

N

t"q e ~ ',,D t'q

c~ 0 D

tt~

t'q

t"q

t"q e¢~

d Z al

,.4t<

r,.)

a~ c~ 0 Z

o

--2

~

<

..,..

II

Z

o 0 0

<

0 0

250

•~

~

~.-g

Z

II

II ~*....~

~

e-

~:~..~

~ ~.~..-. ",~t" ~ -

¢'q ~ -

t,N

I

I

~.-

o

ff

ff

o

o

251

252

FLAVINS AND DERIVATIVES

[35]

TABLE II CHROMATOGRAPHIC AND ELECTROPHORETIC PROPERTIES OF 8-HYDROXYRIBOFLAVIN AND OF MODEL COMPOUNDS a'b Electrophoretic mobility at

Re values in solvent Compound Riboflavin FMN FAD 8-Hydroxyriboflavin 7- Methyl-8-hydroxyalloxazine 8-Hydroxylumiflavin

A

B

C

D

0.48 0 0 0.33 0.87 0.60

0.30 0 0 0.17 0.89 0.57

0.70 --0.48 0.65 0.66

0.33 0.15 -0.22 0.81 0.53

pH 3 ( - 10) +13.5 +9 -6.5

pH 7 (+ 1) +16 +14 +9

Adapted from S. Ghisla and S.G. Mayhew, Eur. J. Bi oc he m . 63, 373 (1976). b The solvent systems were: (A) butanol-ethanol-l.0 M sodium acetate, pH 6 (6 : 3 : 1, v/v/v); (B) buthanol-ethanol-water (6:3: 1, v/v/v); (C) butanol-ethanol-2 N ammonia (6: 2 : 2, v/v/v); (D) butanol-acetic acid-water (4 : 3 : 3, v/v/v). Electrophoretic mobilities are in arbitrary units; a comparison with the mobility of riboflavin from the starting line is given in brackets. (+) Mobility toward the anode; ( - ) mobility toward the cathode.

TABLE III REDOX POTENTIALS OF NORMAL FLAVINS AND 8-HYDROXYFLAVINSa Redox potential (pH) (V)

Compound FMN Isoalloxazine derivative: 7,8,10-Trimethyl7,10-Dimethyl-8-hydroxy-

7-Methyl-8-hydroxy-10-ethyl7-Methyl-8-methoxy-10-ethyl1,3,7,10-Tetramethyl-8-hydroxy-

-0.206(7.17) - 0.227 (7.26)

-0.072(3.26) -0.245(5.88) -0.340(7.09) -0.504(10.03)

-0.132(4.19) -0.271(6.18) -0.392(8.03)

-0.193(5.20) -0.302(6.48) -0.441(9.00)

-0.351(7.21) -0.284(7.27) -0.173(7.21)

a Midpoint redox potentials were measured potentiometrically at 25° with platinum electrodes, and saturated calomel electrode as reference (S.G. Mayhew, unpublished; C.D. Whitfleld private communication). Reductive titrations were carried out with sodium dithionite and oxidative titrations with potassium ferricyanide. The potentials reported are with reference to the standard hydrogen electrode at pH 0.

[36]

COVALENTLY BOUND FLAVINS

253

not as might be expected from the properties of the native enzymes. In particular, the complexes with the modified flavin are fluorescent, while the native enzymes are nonfluorescent. Similarly, the addition of benzoate to native D-amino acid oxidase increases the resolution of its visible abso~tion maximum, while it has the opposite effect on the spectrum of the complex with 8-hydroxy-FAD.

[36] S t r u c t u r e , P r o p e r t i e s , a n d D e t e r m i n a t i o n Covalently Bound Flavins

of

By THOMAS P. SINGER and DALE E. EDMONDSON Arecent article in this series 1 discussed the synthesis and isolation of 8a-substituted flavins and ravin peptides in some detail. To minimize repetition, this chapter summarizes only briefly the structure and properties of 8a-substituted flavins and concentrates instead on two aspects not previously covered: the isolation, structure, and chemical properties of 6-substituted covalently bound flavins and the determination of covalently bound flavins other than 8a-N(3)-histidylflavin derivatives. An analytical procedure for flavin peptides containing an 8a-N(3)-histidylriboflavin linkage has appeared in an earlier volume, 2 but procedures for the analysis of the other three types of covalently bound ravin have not been previously described. Structures and Chemical Properties

8a-N (3)-Histidylflavins This covalently bound flavin has the distinction of having been the first peptide-bound flavin to be discovered and also the first structure to be defined. The histidine moiety is covalently bound to the flavin via the N(3)-imidazole nitrogen to the 8a-methylene group of the flavin moiety, as shown in Fig. 1. Since the elucidation of its structurefl "4 this 8a1 D. E. E d m o n d s o n , W. C. K e n n e y , and T. P. Singer, this series, Vol. 53 [46], p. 449. 2 T. P. Singer, J. Salach, P. H e m m e r i c h , and A. Ehrenberg, this series, Vol. 18, Part B [142], p. 416. a j. Salach, W. H. Walker, T. P. Singer, A. Ehrenberg, P. H e m m e r i c h , S. Ghisla, and U. H a r t m a n n , Eur. J. Biochem. 26, 267 (1972). 4 W. H. Walker, T. P. Singer, S. Ghisla, and P. H e m m e r i c h , Eur. J. Biochem. 26, 279 (1972).

METHODS IN ENZYMOLOGY, VOL. 66

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181966-3