Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase.
Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.
Hederstedt
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L
Maguire
J J
JJ
Waring
A J
AJ
Ohnishi
T
T
D001412:Bacillus subtilis / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D008872:Microwaves; D013385:Succinate Dehydrogenase
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D013385:Succinate Dehydrogenase