Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdBdepends on the WhiD [4Fe-4S] cluster

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. InStreptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results fromin vitroandin vivoexperiments with WhiD fromStreptomyces venezuelae(SvWhiD), which differs fromS. coelicolorWhiD (ScWhiD) only at the C terminus. We observed that, likeScWhiD and other Wbl proteins,SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O(2)and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found thatSvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins ofMycobacterium tuberculosisare known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate thatSvWhiD interacts with domain 4 of the principal sigma factor ofStreptomyces, ?(HrdB)(?(HrdB)(4)). Using MS, we determined the dissociation constant (K-d) for theSvWhiD-?(HrdB)(4)complex as ?0.7 ?m, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8?10 NO molecules per cluster, resulted in dissociation into the separate proteins. TheSvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O(2)and NO whenSvWhiD was bound to ?(HrdB)(4), consistent with protection of the cluster in the complex.