Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.

Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe-2S] cluster accompanies sulfur insertion.

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.

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters.

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.

CD-monitored redox titration of the Rieske Fe-S protein of Rhodobacter sphaeroides: pH dependence of the midpoint potential in isolated bc1 complex and in membranes.

The redox potential of the Rieske Fe-S protein has been investigated using circular dichroism (CD)-spectroscopy. The CD features characteristic of the purified bc1 complex and membranes of Rhodobacter sphaeroides were found in the region between 450 and 550 nm. The difference between reduced and oxidized CD-spectra shows a negative band at about 500 nm with a half of width 30 nm that corresponds to the specific dichroic absorption of the reduced Rieske protein (Fee, J.A. et al. (1984) J. Biol. Chem. 259, 124-133; Degli Esposti, M. et al. (1987) Biochem. J. 241, 285-290; Rich, P.R. and Wiggins, T.E. (1992) Biochem. Soc. Trans. 20, 241S). It was found that the redox potential at pH 7.0 for the Rieske center in the isolated bc1 complex and in chromatophore membranes from the R-26 strain of Rh. sphaeroides is 300 +/- 5 mV. In chromatophores from the BC17C strain of Rh. sphaeroides, the Em value measured for the Rieske iron-sulfur protein (ISP) was higher (315 +/- 5 mV), but the presence of carotenoids made measurement less accurate. The Em varied with pH in the range above pH 7, and the pH dependence was well fit either by one pK at approximately 7.5 in the range of titration, or by two pK values, pK1 = 7.6 and pK2 = 9.8. Similar titrations and pK values were found for the Rieske Fe-S protein in the isolated bc1 complex and membranes from the R-26 strain of Rb. sphaeroides. The results are discussed in the context of the mechanism of quinol oxidation by the bc1 complex, and the role of the iron sulfur protein in formation of a reaction complex at the Qo-site.