| Abstract
| - Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom betweenC6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purifiedrecombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe−2S]2+ clusterper monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of theas-purified [2Fe−2S]-containing BioB with Fe2+ and S2- produces a form of BioB that containsapproximately one [2Fe−2S]2+ and one [4Fe−4S]2+ cluster per monomer ([2Fe−2S]/[4Fe−4S] BioB).In the absence of added Fe, the [2Fe−2S]/[4Fe−4S] BioB is active and can produce up to approximately0.7 equiv of biotin per monomer. To better define the roles of the Fe−S clusters in the BioB reaction,Mössbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the statesof the Fe−S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe−4S]2+ cluster is stable during the reaction and present in the SAM-bound form, supporting the currentconsensus that the functional role of the [4Fe−4S] cluster is to bind SAM and facilitate the reductivecleavage of SAM to generate the catalytically essential 5‘-deoxyadenosyl radical. The results alsodemonstrate that approximately 2/3 of the [2Fe−2S] clusters are degraded by the end of the turnoverexperiment (24 h at 25 °C). A transient species with spectroscopic properties consistent with a [2Fe−2S]+ cluster is observed during turnover, suggesting that the degradation of the [2Fe−2S]2+ cluster isinitiated by reduction of the cluster. This observed degradation of the [2Fe−2S] cluster during biotinformation is consistent with the proposed sacrificial S-donating function of the [2Fe−2S] cluster putforth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry40, 8352−8358). Interestingly,degradation of the [2Fe−2S]2+ cluster was found not to parallel biotin formation. The initial decay rateof the [2Fe−2S]2+ cluster is about 1 order of magnitude faster than the initial formation rate of biotin,indicating that if the [2Fe−2S] cluster is the immediate S donor for biotin synthesis, insertion of S intodethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe−2S] cluster may not be theimmediate S donor. Instead, degradation of the [2Fe−2S] cluster may generate a protein-bound polysulfideor persulfide that serves as the immediate S donor for biotin production.
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