Quinol oxidation by the bc 1 complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q o site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c 1 . Effects of mutations to the ISP that change the redox potential and͞or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or ␤-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H ؉ transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.The bc 1 complex family of enzymes plays a central role in all the main pathways of energy conversion, and the photosynthetic apparatus of Rhodobacter sphaeroides exemplifies one of the simplest of these (1-5). This system is convenient experimentally because of the ease with which electron transfer can be initiated by illumination (6). X-ray crystallographic structures of mitochondrial complexes (7-10) contain at their core the three catalytic subunits, cytochrome (cyt) b, cyt c 1 , and the Rieske iron-sulfur protein (ISP), common to the bacterial enzymes (11-13). Homology models of these show that the catalytic superstructure is highly conserved, as had been expected from studies of the mechanism, which is essentially the same in the two systems (1-5). The bc 1 complex catalyzes the oxidation of quinol and the reduction of cyt c (or c 2 ) through a modified Q cycle (1-6, 14-17). Two separate internal electron transfer chains connect three catalytic sites. At one site, heme c 1 is oxidized by cyt c 2 . Two catalytic sites in cyt b are involved in oxidation or reduction of ubiquinone. In the bifurcated reaction at the quinol-oxidizing site (the Q o site), one electron from quinol is passed to the ISP, which transfers it to cyt c 1 , while the semiquinone produced is oxidized by another chain consisting of the two b hemes of cyt b. At the quinone-reducing site (Q i -site), electrons fr...

Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) cluster.