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8 - ( A - 3 - H I S T I D Y L - ) F L A V I N I N D - 6 - H Y D R O X Y N I C O T I N E O X I D A S E 1 0 7 3

oxidase 3 . Moreover, methylene blue was shown to be a strong inhibitor of the overall reaction. One-electron acceptors are also ineffective with the D-specific enzyme; however, methylene blue and 2.6-dichlorophenol-indophenol are able to reoxidize the reduced D-6-hydroxynicotine oxidase. In the pre-sence of these dyes the rates of the overall process are higher than those with oxygen.

Anaerobic titrations of both enantiozymes with their respective substrates produced a steady tran-

1 K. DECKER and H. BLEEG, Biochim. biophysica Acta [Am-sterdam] 105 ,313 [1965] .

2 M . GLOGER a n d K . DECKER, Z . N a t u r f o r s c h . 2 4 b, 1 0 1 6 [ 1 9 6 9 ] .

sition from the oxidized to the reduced form. Spec-tral evidence of a semiquinoid intermediate stage could not be obtained. This was seen, however, after illumination of the enzymes in the presence of EDTA.

It remains to be elucidated whether and to what extent reactivity towards artificial two-electron ac-ceptors of D- and L-6-hydroxynicotine oxidase and the type of coenzyme binding are a consequence of the respective quaternary structure of these proteins.

3 K. DECKER and V. D. DAI, Europ. J. Biochem. 3, 132 [1967]. 4 V. D. DAI, K. DECKER, and H. SUND, Europ. J. Biochem. 4,

9 5 [ 1 9 6 8 ] .

Covalently Bound Flavin in D-6-Hydroxynicotine Oxidase from Arthrobacter oxidans

M . BRÜHMÜLLER, H . MÖHLER, a n d K . DECKER

Biochemisches Institut der Universität, Freiburg i. Br., West-Germany

(Z . Naturforsch. 27 b, 1073—1074 [1972] ; received May 10, 1972)

Flavoprotein, Covalently bound FAD, 8a- (A3-histidyl) -riboflavin

D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95 ° C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8a-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8a-(A-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125 ° C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a-(A-3-histidyl)-flavin-adenine-dinucleotide in the active center.

Unusual Abbreviations: HNO-flavin, riboflavin attached to one amino acid of D-6-hydroxynicotine oxidase; His-ribo-flavin, 8a-histidyl-riboflavin.

D-6-Hydroxynicotine oxidase from Arthrobacter oxidans, catalyzing the oxidative cleavage of D-6-hydroxynicotine to (6-hydroxypyridyl-(3) )-(y-N-methyaminopropyl)-ketone, contains one mole of FAD per mole of enzyme. Methods known to rever-sibly dissociate the flavin coenzyme from the apo-protein failed to release the coenzyme. Treatment of the enzyme with 5% trichloroacetic acid resulted in a supernatant without any absorbance in the visible range whereas the precipitate displayed the typical flavin spectrum. Tryptic digestion of the

Requests for reprints should be sent to Prof. Dr. K. DECKER, Biochemisches Institut der Universität, D-7800 Freiburg, Hermann-Herder-Str. 7.

purified enzyme yielded a fluorescent product, migrating differently than FAD in paper chromato-graphy and high voltage electrophoresis. These re-sults were taken as evidence for the presence of a covalently bound flavin in D-6-hydroxynicotine oxi-dase.

To identify the covalent linkage between FAD and the apoprotein, an amino acid derivative of riboflavin (HNO-flavin) was isolated from flavin peptides either by hydrolysis in 6 N HCl at 95 ° C in vacuo for 12 h 1 or with aminopeptidase M con-taining prolinase. The aminopeptidase digest was then incubated in 10% trichloroacetic acid in order to hydrolyze the flavin dinucleotide to the mono-nucleotide and subsequently at pH 5.7 with acid phosphatase for dephosphorylation. The resulting HNO-flavin was purified by preparative thin layer

1 0 7 4 8 - ( A - 3 - H I S T I D Y L - ) F L A V I N I N D - 6 - H Y D R O X Y N I C O T I N E O X I D S E

chromatography on cellulose with ri-butanol-acetic acid-H20 2 : 1 : 2 and 8 : 1 : 91, respectively. It showed the following characteristics:

Compared to the spectrum of riboflavin, HNO-flavin showed at pH 7 a hypsochromic shift of the peak at 370 nm to 346 nm. It was further displaced to 341 nm at pH 2 .2 ; this shift reflects an additio-nal protonation of the amino acid. In 6 N HCl the absorption band of riboflavin at 395 nm was dis-placed to 370 nm in HNO-flavin. This spectral be-haviour is characteristic for 8a-substitution of the flavin nucleus 2 - 4 .

The fluorescence intensity of HNO-flavin was pH-dependent. The maximal intensity at pH 3.2 was quenched by 86% at pH 7.0. The pK of fluorescence quenching was 4.65. This suggests a secondary or tertiary amino group attached to the flavin5. For synthetic 8a-histidyl-riboflavin, containing predomi-nantly the ^-3-isomer, a pK of 4.7 was found.

Hydrolysis of HNO-flavin in 6N HCl at 125 °C for 20 h resulted in the release of histidine, glutamic and aspartic acid. Glutamate and aspartate as com-ponents of HNO-flavin are excluded by the fluores-cence data. The release of 1 mole of histidine per mole of HNO-flavin in 80% yield is in agreement with all the data presented for HNO-flavin.

1 W . H . WALKER a n d T . P . SINGER, J . b i o l . C h e m i s t r y 2 4 5 , 4 2 2 4 [ 1 9 7 0 ] .

2 P . HEMMERICH, A . EHRENBERG, W . H . WALKER, L . E . G . ERIKSON, J. SALACH, P . BADER, a n d T . P . SINGER, F E B S L e t t . 3 , 3 7 [ 1 9 6 9 ] .

Table. Characteristics of HNO-flavin and Histidyl-riboflavin.

Properties HNO-flavin His-riboflavin

A b s o r p t i o n spectrum in 370 n m , 370 n m , 6 N HCl ( A m a x ) 265 n m 265 n m Fluorescence -quenching at p H 7 as c o m p a r e d t o p H 3.2 8 6 % 9 4 %

p K o f fluorescence-quenching 4 .65 4 .7

nmo les histidine per n m o l e l iberated b y ac id hydro lys is 0.8 0 .6 RP o f iV-3-isomer 0.27 0 .28 RF o f jV- l - i somer — 0.33

HNO-flavin, obtained with aminopeptidase, mi-grated identically with the iV-3-isomer of synthetic 8a-histidyl-riboflavin in thin layer chromatography on cellulose with n-butanol-acetic acid-H20 (2 :1 :2 ) .

A comparison of synthetic 8a-histidyl-flavin and HNO-flavin is given in the Table. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme con-tains 8a- (7V-3-histidyl) -flavin-adenin-dinucleotide in the active center.

The authors are most grateful to Professor P. HEM-MERICH and Dr. S. GISHLA for the supply of synthetic 8a-histidyl-riboflavin.

3 R . HENDRIKS a n d J. R . CRONIN, B i o c h i m . b i o p h y s i c . R e s . C o m m u n . 4 4 , 3 1 3 [ 1 9 7 1 ] .

4 S . GHISLA a n d P . HEMMERICH, F E B S L e t t . 1 6 , 2 2 9 [ 1 9 7 1 ] . 5 S . GHISLA, U . HARTMANN, a n d P . HEMMERICH, A n g e w . C h e

m i e 8 2 , 6 6 9 [ 1 9 7 0 ] .