Studies on succinate dehydrogenase : site of attachment of the covalently-bound flavin to the peptide chain

Abstract

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. SDflavin 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 spectrometry 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.