Histidyl-Flavins

AFOO-Phosphorylhistidine AT -f Methyl)histidine 4-(iodo)histidine 8a-(histidyl)flavin ADP-ribosylation... 

As a result of collaborative work among the laboratories of Ehren-berg, Hemmerich, and Singer 161-163), the structure of the bound flavin was elucidated to be 8 -(A(-3-histidyl) flavin adenine dinucleotide. The structure of the histidyl flavin and the sequence of a flavin-bound penta-peptide isolated by proteolytic digestion are shown in Fig. 28. Singer et al. 23,25) have discussed the details of these studies. 

Walker, W. H., and Singer,T. P., 1970, Identification of the covalently bound flavin of succinate dehydrogenase as 8-alpha-(histidyl) flavin adenine dinucleotide. J. Biol. Chem. 245 4224n4225. 

The absorption spectra of 8a-histidyl-flavins (9) are, in their general features, very similar to those of unmodified flavins, e.g. FAD, FMN or lumiflavin 186). In the neutral state (pH 7), the lowest energy band at 445 nm (s 12.000) and the UV band at 268 nm are unaltered, whereas the second band exhibits a hypsochromic shift to 355 nm as compared to riboflavin (372 nm). Upon protonation of the histidine moiety (pK 4.7) the same band is shifted further to 345 nm. In the SD-flavin cation (protonation at N(l)) the band centered at 400 nm in normal flavins, is now partially resolved and has a maximum at 370 nm and a pronounced shoulder at 412 nm. 

The most characteristic feature of 8a-histidyl flavins however, is... 

The covalent 8a-N(3)-histidyl FAD of mitochondrial succinate dehydrogenase functions as a two-electron acceptor in the oxidation of succinate to fumarate and as a one-electron donor in the reduction of the iron-sulfur centers of the enzyme. Recent ESR spectroscopic data have shown the covalent flavin semiquinone... 

Mitochondrial succinate dehydrogenase, which catalyzes the reaction of Eq. 15-21, contains a flavin prosthetic group that is covalently attached to a histidine side chain. This modified FAD was isolated and identified as 8a-(Ne2-histidyl)-FAD 219 The same prosthetic group has also been found in several other dehydrogenases.220 It was the first identified member of a series of modified FAD or riboflavin 5 -phosphate derivatives that are attached by covalent bonds to the active sites of more than 20 different enzymes.219... 

Fia. 28. Structure of histidyl-8-o-FAD (left) and the sequence of the flavin penta-peptide (right) of succinate dehydrogenase. From Singer et al. (SB). 

The second modified flavin of natural origin to be discovered was 8a-S-cysteinyl-FAD, the coenzyme of monoamino oxidase from liver and kidney outer mitochondrial membranes. Taking their departure from investigations of Yasunobu (8J) and Hellerman (SO), which indicated the presence of covalently bound flavin in preparations of this enzyme, Singer and his group (85, 185) isolated the flavinyl peptide by degradation of MAO with trypsin-chymotrypsin and identified cysteine as the amino acid residue bound next to the flavin moiety (184). The absorption spectrum of the flavin peptide from monoamino oxidase is readily differentiated from that of riboflavin by a hypsochromic shift of the second absorption band (360 nm, compare with 372 for riboflavin), in the neutral oxidized state (44, 184). It is similar to that of 8a-histidyl-riboflavin in the cationic state in that the band centered around 400 nm (abs. max. 375 nm, shoulder at 410 nm) is partially resolved. The fluorescence emission (4, 30) is only 10% of that of riboflavin, but oxidation with peracids raises it to 90% of riboflavin emission. 

The chemical properties of 8a-cysteinyl-riboflavin (12) closely resemble those of activated thioethers and have been investigated mainly with model compounds carrying n-alkyl derivatives in position 8p 44). Reaction of (13), for example, with peracids leads to the sulfones (14), which in turn can be cleaved by reduction with dithionite to yield unsubstituted flavin and the corresponding sulfinate (25). Whereas the flavin C(8a)/histidyl-N(3) bond in (9) can be reduced only in rather... 

Most recent investigations by Edmondson and Kenney (Biochem. Biophys. Res. Comm. 68, 242 [1976]) have shown, that an assignment made on p. 494 is incorrect Compound 9 b, obtained upon prolonged heating of SD-flavin (natural or synthetic) under acid conditions (9a), is not the N(l)-histidyl-isomer of 9a, but a derivative of 9a modified in the ribityl side chain. The true N(l)-histidyl-isomer is obtained synthetically as a byproduct of the synthesis of 9 a and has been identified with the covalently bound flavoprotein from (3-cyclopiazonate oxidocyclase. 

Mohler, H., M. Bruhmuller, and K. Decker Covalently Bound Flavin in D-6-Hydroxynicotine Oxidase from Arthrobacter oxidans. Identification of the 8a-(N-3-Histidyl)-riboflavin-Linkage between FAD and Apoenzyme. Eur. J. Biochem. 29, 152 (1972). 

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