Studies zyxwv on Succinate Dehydrogenase Site of Attachment of the Covalently-Bound Flavin to the Peptide Chain Jim SALACH, Wolfram H. WALKER, and Thomas P. SINGER Division of Molecular Biology, Veterans Administration Hospital and Department of Biochemistry and Biophysics, University of California, San Francisco Anders EHRENBERG Biofysiska Institutionen, Stockholm Universitetet, and Medicinska Nobelinstitutet, Karolinska Institutet, Stockholm Peter HEMMERICH, Sandro GHISLA, and Ursula HARTMANN Fachbereich Biologie, Universitat Konstanz (Received June 2/0ctober 15,1971) Improved methods have been devised for the isolation in pmole quantities of a pure flavin pentapeptide and its acid-hydrolysis product (SD-flavin) from inner-membrane preparations of heart mitochondria and from soluble, purified succinate dehydrogenase. SD-flavin differs from riboflavin in still having an amino acid covalently linked to the isoalloxazine ring system. SD- flavin may be compared with riboflavin and with various 8n-substituted synthetic flavins by optical spectrophotometry in the neutral and cationic states and by ESR and ENDOR spectro- metry in the cationic radical state. On the basis of these experiments is was concluded that the FAD prosthetic group of mitochondria1 succinate dehydrogenase is covalently linked through the 8n-position to the peptide backbone of the protein. This conclusion is in accord with the acid stability of the natural product and its tendency to yield riboflavin under reductive conditions. The unusual pH-fluorescence spectrum of the flavin strongly suggests that the 8n-methylene group is linked to an amino acid through a tertiary nitrogen group. Early in 1950 the suggestion arose that, in addi- tion to riboflavin, FMN and FAD, a fourth form of flavin exists in nature, one which is not extracted from tissues by conventional denaturation methods but requires proteolytic digestion for extraction [1,2]. Unmistakable evidence that this new form of riboflavin, which subsequently became known as “bound flavin” or “covalently bound flevin” originates from succinate dehydrogenase had to await the first isolation of the enzyme in soluble, zyxwvu Abbreviations and Definitions. ESR, electron-spin res- onance zyxwvutsrqp ; ENDOR, electron nuclear double resonance ; NMR, nuclear magnetic resonance; SD-flavin, acid-hydro- lysis product of a flavin pentapeptide isolated from inner membrane preparations of heart mitochondria; non-phos- phorylating preparation (ETP in other publications), a non-phosphorylating inner-membrane preparation from beef- heart mitochondria ; phosphorylating preparation (ETPH in other publications), a phosphorylating inner-membrane preparation from beef-heart mitochondria. Enzymes. Succinate dehydrogenase, or succinate : ac- ceptor oxidoreductase (EC 1.3.99.1); trypsin (EC zyxwvut 3.4.4.4); chymotrypsin (EC 3.4.4.5). zyxwvutsr 18. purified form [3]. Shortly thereafter Kearney and Singer [4,5] presented evidence that succinate de- hydrogenase contained covalently-linked FAD and that on digestion with trypsin and chymotrypsin a flavin peptide with markedly-different properties from known flavins could be isolated. These conclu- sions were confirmed and extended by Wang et zy al. [S]. Subsequent studies [7,8] disclosed that SD- flavin differs from previously-known flavins in show- ing a hypsochromic shift of its 375-nm absorption band to 345-350 nm in the neutral oxidized state, inactivity in the D-amino acid oxidase test at the FAD level, greater water solubility, failure to yield free authentic l u d a v i n on alkaline irradiation, and a characteristic pH-fluorescence curve which shows a maximum a t pH 3.2 to 3.4 with a pK of 4.5 & 0.1, and no significant fluorescence a t neutral pH. Kearney [8] also showed that hydrolysis a t 95 “C in 6-N HC1 yields a flavin which was different from all previous-known flavins, particularly ribo- Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/5520/ URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-55209 First publ. in: European Journal of Biochemistry 26 (1972), pp. 267-278