Origins of specificity and cross‐talk in metal ion sensing by B acillus subtilis Fur

Summary Fur ( f erric u ptake r egulator) is the master regulator of iron homeostasis in many bacteria, but how it responds specifically to Fe ( II ) in vivo is not clear. Biochemical analyses of B acillus subtilis Fur ( BsFur ) reveal that in addition to Fe ( II ), both Z n( II ) and Mn ( II ) allosterically activate BsFur – DNA binding. Dimeric BsFur co‐purifies with site 1 structural Zn ( II ) ( Fur 2 Zn 2 ) and can bind four additional Zn ( II ) or Mn ( II ) ions per dimer. Metal ion binding at previously described site 3 occurs with highest affinity, but the Fur 2 Zn 2 : Me 2 form has only a modest increase in DNA binding affinity (approximately sevenfold). Metallation of site 2 ( Fur 2 Zn 2 : Me 4 ) leads to a ∼ 150‐fold further enhancement in DNA binding affinity. Fe ( II ) binding studies indicate that BsFur buffers the intracellular F e( II ) concentration at ∼ 1 μ M . Both Mn ( II ) and Zn ( II ) are normally buffered at levels insufficient for metallation of BsFur site 2, thereby accounting for the lack of cross‐talk observed in vivo . However, in a perR mutant, where the BsFur concentration is elevated, BsFur may now use Mn ( II ) as a co‐repressor and inappropriately repress iron uptake. Since PerR repression of fur is enhanced by Mn ( II ), and antagonized by Fe ( II ), PerR may co‐regulate F e( II ) homeostasis by modulating BsFur levels in response to the Mn ( II )/ Fe ( II ) ratio.