The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from and experiments with WhiD from (WhiD), which differs from WhiD (WhiD) only at the C terminus. We observed that, like WhiD and other Wbl proteins, WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that WhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of ... More
The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from and experiments with WhiD from (WhiD), which differs from WhiD (WhiD) only at the C terminus. We observed that, like WhiD and other Wbl proteins, WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that WhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that WhiD interacts with domain 4 of the principal sigma factor of , σ (σ). Using MS, we determined the dissociation constant ( ) for the WhiD-σ complex as ∼0.7 μm, 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. The WhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O and NO when WhiD was bound to σ, consistent with protection of the cluster in the complex.
