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

Abstract

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.

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

“…Thus, there is a possibility that there is a minor contribution to the relief of power saturation from relaxation enhancement of S-1 red by S-3 red through a putative magnetic interaction (52,53). There is precedent for an interaction between the oxidized S-3 center (S T ϭ 1/2) and the reduced S-1 center in Micrococcus luteus (54) and B. subtilis (55). Taken together, the minor relief of power saturation of the succinate-reduced S-3 center upon reduction of the sample with dithionite (Fig.…”

Processing of Bacillus subtilis succinate dehydrogenase and cytochrome &‐558 polypeptides

“…The Fp in B. subtilis undergoes at least two posttranslational modifications before assembly into the SDH complex; the N-terminal Met is removed (this work) and the His at position 40 is flavinylated [25]. To determine if these two modifications also occur in E. coli we isolated fig.3).…”

“…To determine if these two modifications also occur in E. coli we isolated fig.3). Furthermore, they show that the Fp from the two bacteria is processed identically at the Nterminus which contains the&$ fold [9,26] [21,25]. Folding of apo-Fp seems necessary before the cofactor can be bound; e.g.…”

“…Different B. subtilis mutant Fp subunits which lack flavin as a result of single amino acid substitutions co-migrate or migrate slower than the wild-type subunit ( [21] and Hederstedt, unpublished). Second, the SDHcytochrome b-558 complex with a full complement of iron-sulfur centers can be assembled in B. subtilis without the flavin [25,30]. Third, the ironsulfur center S-l is not detectable in the B. subtilis SDH protein present in E. coli (pSH1047) cell lysates (Andersson and Hederstedt, preliminary EPR data).…”

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex

“…The homologies increase to 53 and 57% (64% between themselves) when pairs scoring ¢0.1 in the MDM78 matrix (49) are included. There is a particularly high degree of sequence conservation in the N-terminal regions, which contain the PA-aA-PB segments that contact the bottom of the AMP portion of the FAD in the FAD-binding folds (34,60) (Fig. 5).…”