COVALENT STRUCTURE OF THE FLAVOPROTEIN SUBUNIT OF THE FLAVOCYTOCHROME-C - SULFIDE DEHYDROGENASE FROM THE PURPLE PHOTOTROPHIC BACTERIUM CHROMATIUM-VINOSUM

The amino acid sequence of the flavoprotein subunit of Chromatium vino sum flavocytochrome c-sulfide dehydrogenase (FCSD) was determined by a utomated Edman degradation and mass spectrometry in conjunction with t he three-dimensional structure determination (Chen Z et al., 1994, Sci ence 266:430-432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 +/- 44 Da measured by LDMS. There are six cysteine r esidues, among which Cys 42 provides the site of covalent attachment o f the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the F AD. The flavoprotein subunit of FCSD is most closely related to glutat hione reductase (GR) in three-dimensional structure and, like that pro tein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in seq uence is not significantly greater than for random comparisons. The fi rst domain binds FAD in both proteins. Domain 2 of GR is the site of N ADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfu r compounds. Domains 1 and 2 of FCSD, as of CR, are homologous to one another and represent an ancient gene doubling. The third domain provi des the dimerization interface for GR, but is the site of binding of t he cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite ad duct formation and decay, have now been identified from the three-dime nsional structure and the sequence as Cys 161/Cys 337 disulfide, Trp 3 91, Glu 167, and the positive end of a helix dipole.