Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

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Abstract

WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster, and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single [4Fe-4S](2+) cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and pH 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. [4Fe-4S](2+) WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apoprotein destabilized the protein fold significantly but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay, demonstrating that it does not function as a general protein disulfide reductase.

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