Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse-chase and immunoprecipitation exper iments showed that Irr degraded in response to 6 mu M iron with a half-life of approximate to 30 min and that this regulated stability was the princip al determinant of control by iron. Irr contains a heme regulatory motif (HR M) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron, Ad dition of heme to low iron (0.3 mu M) cultures was sufficient for the disap pearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein b ound heme with high affinity and caused a blue shift in the absorption spec trum of heme to a shorter wavelength. A Cys(29) -->Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and t he spectral blue shift. In vivo turnover experiments showed that, unlike wi ld-type Irr, IrrC29A was stable in the presence of iron. We conclude that i ron-dependent degradation of Irr involves direct binding of heme to the pro tein at the HRM. The findings implicate a regulatory role for heme in prote in degradation and provide direct evidence for a functional HRM in a prokar yote.