Figure 6:

(A) EPR spectrum of BioB prior to and during a single turnover of biotin production. BioB (100 μM monomer) was generated with 1:1 [2Fe-2S]²⁺ and [4Fe-4S]²⁺ clusters and was preincubated with excess substrates in the presence or absence of FeCl₃, Na₂S, and DTT, and samples (∼200 μL) were withdrawn for EPR analysis. The top curves show that no signal is observed in the absence of enzyme or dethiobiotin. In the presence of FeCl₃, Na₂S, and DTT the time-dependent formation of a new EPR-active species is observed. This species is not observed in the absence of FeCl₃, Na₂S, and DTT. Biotin (∼0.9 equiv per monomer) is formed under both conditions. (B) EPR spectrum of BioB after a single turnover and following chemical reduction. BioB with 1:1 [2Fe-2S]²⁺ and [4Fe-4S]²⁺ clusters was incubated with the substrates FeCl₃, Na₂S, and DTT for 120 min and then repurified by anaerobic gel filtration chromatography, resulting in ca. 2-fold dilution. The EPR spectrum is otherwise identical to the final spectrum in panel A. The protein was then reduced by incubation with dithionite (2 mM) for 30 min. The final reduced spectrum resembles the EPR spectrum previously reported for reductively reconstituted [4Fe-4S]⁺ enzyme (20), shown as a dashed curve scaled for comparison. The small resonance at g = 2.05 is not observed in all enzyme preparations and may be due to a contaminating protein or metal.