Succinate Dhydrogenase Mutants of Bacillus Suybtilis Lacking Covalently Bound Flavin in the Flavoprotien Subunit

Abstract

Succinate dehydrogenase consists of two unequal subunits; Fp and Ip. An FAD group is covalently linked to a histidyl residue in the Fp subunit. The mechanism by which flavin is attached to protein is not known. Covalently bound flavin was studied in wild-type and succinate-dehydrogenase-negative Bacillus subtilis. The Fp subunit of succinate dehydrogenase was found to be the only (major) flavinylated protein in the cell. Mutants lacking covalently bound flavin and still containing the Fp polypeptide are described. It is shown that the flavin is not essential for assembly and membrane binding of succinate dehydrogenase in B. suhtilis.

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

“…Four mutant strains of Bacillus subtilis that lack SDH activity have been isolated by Hederstedt (1983). The mutants assemble correctly in the membrane, possess intact Fe-S clusters, but are totally devoid of FAD.…”

“…The flavoCovalent attachment of f2avins to enzymes 15 protein domains of SDH and FRD contain the active sites of the enzymes, and are highly conserved at the amino acid level around the sites of flavin attachment. Initially, this was taken to indicate that formation of the covalent linkage with FAD is mediated by an auxiliary enzyme specific for this potential recognition sequence, but, in light of recent work, this now seems improbable.Four mutant strains of Bacillus subtilis that lack SDH activity have been isolated by Hederstedt (1983). The mutants assemble correctly in the membrane, possess intact Fe-S clusters, but are totally devoid of FAD.…”

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

“…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.…”

“…First, the increased electrophoretic mobility of Fp isolated from E. coli is not unambiguously explained by the absence of flavin. 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of single amino acid substitutions co-migrate or migrate slower than the wild-type subunit ( [21] and Hederstedt, unpublished).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). Similarly, mutants of B. subtilis mutated in Fp such that SDH is not assembled also lack flavin in Fp as well as center S-l [21,30].…”