Role of the [2Fe−2S] Cluster in Recombinant Escherichia coli Biotin Synthase

Abstract

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe−2S]²⁺ cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe−2S]-containing BioB with Fe²⁺ and S^2- produces a form of BioB that contains approximately 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 approximately 0.7 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 states of 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 current consensus that the functional role of the [4Fe−4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5‘-deoxyadenosyl radical. The results also demonstrate that approximately ²/₃ of the [2Fe−2S] clusters are degraded by the end of the turnover experiment (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 is initiated by reduction of the cluster. This observed degradation of the [2Fe−2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe−2S] cluster put forth 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 rate of 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 into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe−2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe−2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.