Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme.
Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D013045:Species Specificity; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D013385:Succinate Dehydrogenase