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FLAVIN-SENSITIZED PHOTOOXIDATIONS 1035

11 W. HAAS, Dissertation, Universität Konstanz 1972. 1 2 M . B . K A T A N , L . J . GILING, a n d J . D . W . V A N V O O R S T ,

Biochim. biophysica Acta [Amsterdam] 234, 242 [1971]. 1 3 P . HEMMERICH, i n : R . S . HARRIS , P . L . M U N S O N , a n d E .

DICZFALUSY (Editors), Vitamins and Hormons, Vol. 28, p. 467, Academic Press, New York 1970.

1 4 T . W A L S H , A . S C H O N B R U N N , a n d R . H . ABELES, J . b i o l . Chemistry 246, 6855 [1971].

15 P. HEMMERICH, in: 14. Mosbacher Colloquium der Gesell-schaft für Physiologische Chemie, p. 183, Springer, Heidel-berg 1963.

1 6 A . D E K O K , C . VEEGER, a n d P . HEMMERICH, i n : H . K A M I N (Editor), Flavin and Flavoproteins, p. 63, University Park Press, Baltimore 1971.

1 7 W . - R . KNAPPE a n d P . HEMMERICH, F E B S - L e t t e r s 1 3 , 2 9 3 [1971],

18 M. BRÜSTLEIN, Dissertation, Universität Konstanz 1971. 1 9 M . BRÜSTLEIN a n d P . HEMMERICH, F E B S - L e t t e r s 1 , 3 3 5

[1968].

pH-Dependence, Isotope Effect and Products of Flavin-sensitized Photodecarboxylation and Photodehydrogenation

W . HAAS and P. HEMMERICH

Department of Biology, University of Konstanz, Germany

(Z. Naturforsch. 27 b, 1035—1037 [1972] ; received May 10, 1972)

Flavin-sensitized photodecarboxylation and photodehydrogenation

The pH-dependence of flavin-sensitized photodehydrogenation and oxidative photodecarboxyla-tion shows a change of reaction rate with an apparent pK of about 5.5. This "photo-pK" cannot be assigned to any known ground state or excited flavin or substrate species, nor is it related to the kind of bond being broken (C —H or C —COO) . Therefore, it is attributed to the pK of a covalent flavin-substrate intermediate. Carboxylates R —COO - react by decarboxylation, the nature of R determining only the rate of reaction. The acids R —COOH themselves are unreactive, unless functionally substituted at the a-carbon. Hence X—CH(R)—COOH behaves as does X—CH2 —R,

+ + for X = O H , OR, NH2 , NR2 . R3N—CH(R) — COO" is unreactive, while H S N - C H ( R ) - C O O " is slowly dehydrogenated through its neutral tautomer which is favored in the flavin-substrate exciplex.

Photooxidations sensitized by flavoquinone (1) show a change of reaction rate between pH 4 and 7 1 _ 5 . This change, observed with numerous sub-strates, is sigmoid with an apparent pK of about 5.5 (Fig. 1 ) . This "photo-pK" cannot be attributed to a substrate pK nor to the pK of flavin species in any known redox 5 or even photoexcited7 ' 8 state. The "photo-pK" cannot be attributed to flavin-sub-strate JT-complexes since it is independent of sub-strate structure and the kind of bond being broken (C — H or C — C O O " ) . Hence we assign the "photo-p K " to a short-lived, covalent flavin-substrate inter-mediate as could be trapped and characterized in the case of phenylacetate (R = H) under alkaline conditions9 (2, Scheme I ) . The reaction pathway via 2 is not obligatory, since some substrates of flavoquinone-sensitized photooxidations show no "photo-pK" (Fig. 2 ) . Furthermore, kinetic and iso-tope-exchange data1 0 demonstrate a second, direct

Requests for reprints should be sent to Prof. Dr. P. HEM-MERICH, Fachbereich Biologie der Universität, D-7750 Konstanz, Germany.

pathway for the formation of 3 (R = H ) . The sub-strate residue at position C ( 8 ) in the cyclohexa-2,5-dien-l-imine type intermediate 2 readily migrates in an acid-catalyzed rearrangement to position N ( 5 ) yielding the well known 5-alkyl-l,5-dihydro-flavin (3) and subsequently the thermodynamically more stable 4a-alkyl-4a,5-dihydro-flavin (4 ) by ther-mal rearrangement n .

In the case of mandelate (R = OH) only the final 1,5-dihydroflavin (5) can be trapped. W e propose from the pH-dependence of the reaction rate showing the "photo-pK" = 5.5, that in this case a species of type 2 is also formed but decays very rapidly with liberation of benzaldehyde. The latter has been characterized quantitatively.

From this it seems that flavoquinone has two "photoactive sites", i.e. 4a,5-C = N and 8,9-C = C bonds. The choice between them depends on the nature of the substrate and on reaction conditions (pH, temperature and solvent polarity) which in-fluence the structure of non-covalent "preequili-brium complexes". These are typical "exciplexes"

1036 W . H A A S A N D P . H E M M E R I C H

/arbitrary \ units

initial rate

(a r b i t r a r y ̂

k un i t s >

2-

CHj-CH^S-CHj

i * t> 8 pH

Fig. 2. pH-dependence of anaerobic flavin photoreduction by different substrates (0.01 M) in 50% aqueous methanol. Other

conditions are the same as in Fig. 1.

R=H,CH3

Fig. 1. pH-dependence of anaerobic flavin photo-reduction. Initial rates, measured by the decrease of the long wavelength flavin absorption band, are plotted against pH. Substrate concentration was 0.1 M, 3-methyl-lumiflavin 7 x 10~ 5 M, NaC104 0.1 M, buffers 0.01 M (sulfate < pH 3 < acetate < p H 6 < phosphate). Illumination by tungsten lamp with wavelengths 420 < A (nm) < 500 (inter-ference filter Balzer K 2) light intensity 105 Lux, temperature 25 °C. — An analogous pH-depen-dence of flavin photoreduction between pH 4 and 7 is found with the following substrates:

C H 3 - S - ( C H , ) 3 - N H 3 + , C H 3 - S - (CH2) 2 -CH (NH2) - C O O H , CH 3 -C (CH3) ÖCH 3 -COO-, C 6 H 5 -CH(OH) -COO" , C 6 H 5 -CH (CH 3 ) -COO".

The pKa-values in the Fig. refer to the various substrate acids and are partially overlapping the

more constant "photo-pK" of ÄS 5.5.

in the case of photoreactions. In enzymes, the apo-protein will influence the site of substrate attack at the flavin.

Quite generally we can show that carboxylic acid anions react with flavoquinone triplet by decarb-oxylation, not dehydrogenation. Accordingly, simi-lar to the phenylacetate reaction, no kinetic isotope effect could be found with a-D-mandelate, in con-trast to the results of PENZER at al. 5. Furthermore, the benzyl groups of the alkylated dihydroflavins and the benzaldehyde formed in these photoreduc-tions show full isotope retention of the starting CH- or CD-equivalents. Carboxylic acid anions without an a-CH-group, like 2,2-dimethyl-pro-pionate and 2-phenyl-isobutyrate, react in the same way as carboxylic acid anions with an a-CH-group. They also show the "photo-pK" and form 5- and 4a-alkylated dihydro-flavins (3 and 4, see Scheme I) . With 2-phenyl-isobutyrate 5 —4a-rearrangement (see Scheme I) proved to be reversible, dependent on pH.

In the case of amino acids the following schema applies:

H / I

HoN —CR —COOH ; "

Flox.h»-1

"Fl-exciplex"

1 R —CO —COOH + N H 3 , slow

H 3 N - C H R - C O O - H 2 N - C H R - C O O " 3 + H+ J

F l o x . h v

Flo* , In -

no reaction 1

R - C H O + CO, + N H , . fa-t

FLAVIN-SENSITIZED PHOTOOXIDATIONS 1037

H , 0 « 3

2 2 0

hv

+ P h - C - C O O l R->

Rl = R 2 = H s pH>7

R 1

C O .

H

3 o

( H

3 R2"<r"Ri Ph

R]= "2=

O H "

C K J -

CH3

R^H.R^OH

^ P h C H O

CHJ

H

A R2-?-r, P h

Scheme I. Reaction pathways of flavin photoreduction by carboxylic acid anions.

The amino acid anions react via decarboxylation, similar to carboxylic acid anions. At basic pH values no keto acids are formed 12 '13 . With phe-nylglycinate no isotope exchange is observed in the benzaldehyde which is formed quantitatively 1. The neutral species reacts by dehydrogenation, but much slower than the anion. The formation of keto acid could be demonstrated in the case of EDTA 13. As indicated by the lack of reactivity of betaine, this is due to the fact that only the neutral and not the di-polar species is active and is obviously stabilized in the flavin-amino acid exciplex.

With acids of type R - CH2 - COO - we find the following sequence of increasing relative reaction rate with flavoquinone triplet:

R = C N ~ N ( C H S ) 3 - CL < O C O C H 3 . < O C H 3 ~ C H 3 ~ N H C O C H 3 < C H =

< N ( C H 3 ) 2 ~ N H 2 .

. O H ~ H

C H 2 C G H 5

We find that decarboxylation prevails over dehydro-genation if a COO_-group, but not if a COOH- or COOR-group is present.

It is concluded that flavin-dependent dehydro-genation and oxidative decarboxylation generally occur through covalent flavin-substrate intermediates of type R — Fl — H which, in the case of most "natu-ral" residues R, decay very readily with formation of R+ (or solvolyses products thereof) and FlredH_

(leucoflavin anion). We want to thank Miss U. H A R T M A N N for technical

assistance.

G . R . PENZER a n d G . K . R A D D D A , B i o c h e m . J . 1 0 9 , 2 5 9 [1968]. P. HEMMERICH, Flavins and Flavoproteins, p. 53, (H. KAMIN), University Park Press, London 1971. D. 0 . CARR and D. E. METZLER, Biochim. biophysica Acta [ A m s t e r d a m ] 2 0 5 , 6 3 [ 1 9 7 0 ] .

G . D . WEATHERBY a n d D . O . C A R R , B i o c h e m i s t r y 9 , 3 4 4 [1970]. G . R . PENZER, G . K . R A D D A , J . A . T A Y L O R , a n d M . B . TAYLOR, Vitamins and Hormones, Vol. 28, p. 441, (R. S. HARRIS, P . L . MUNSON, a n d E . DICZIFALUSY) , A c a d . P r e s s , New York 1970. P . HEMMERICH, in 5 , p . 4 7 0 . J . - M . LHOSTE, A . H A U G , a n d P . HEMMERICH, B i o c h e m i s t r y 5,3290 [1966].

8 J.-M. LHOSTE, Proceedings of the First European Bio physics Congress, p . 221, (E. BRODA, A . LOCKER U. H SPRINGER-LEDERER) , Verlag Wiener Med. Acad., Wien 1971.

9 M . BRÜSTLEIN, W . - R . KNAPPE, a n d P . HEMMERICH, A n g e w Chem. 83, 854 [1971].

10 M. BRÜSTLEIN, 1971, W . HAAS, 1972, Dissertation, Univer sität Konstanz.

1 1 W . H . WALKER, P . HEMMERICH, a n d V . MASSEY, E u r . J Biochem. 13, 258 [1970].

1 2 W . R . FRISELL, C . H . CHUNG, a n d C . G . MACKENZIE, J Biol. Chemistry 234, 1927 [1959].

13 K. ENNS and W. BURGESS, J. Amer. chem. Soc. 87, 5766 [1965].