Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein.

The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stable protein that degrades in response to cellular iron availability. This turnover is heme-dependent, and rapid degradation involves heme binding to a heme regulatory motif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST) when fused to it. Analysis of Irr-GST derivatives with C-terminal truncations of Irr implicated a second region necessary for degradation, other than the HRM, and showed that the HRM was not sufficient to confer instability on GST. The HRM-defective mutant IrrC29A degraded in the presence of iron but much more slowly than the wild-type protein. This slow turnover was heme-dependent, as discerned by the stability of Irr in a heme-defective mutant strain. Whereas the HRM of purified recombinant Irr binds ferric (oxidized) heme, a second site that binds ferrous (reduced) heme was identified based on spectral analysis of truncation and substitution mutants. A mutant in which histidines 117-119 were changed to alanines severely diminished ferrous, but not ferric, heme binding. Introduction of these substitutions in an Irr-GST fusion stabilized the protein in vivo in the presence of iron. We conclude that normal iron-dependent Irr degradation involves two heme binding sites and that both redox states of heme are required for rapid turnover.