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 experiments showed that Irr degraded in response to 6 microM iron with a half-life of approximately 30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) 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. Addition of heme to low iron (0.3 microM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum 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 the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.
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Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Hamza
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O'Brian
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D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D006418:Heme / Q000378:metabolism*; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D014157:Transcription Factors; C114259:iron response regulator protein, Bacteria; D006418:Heme; D007501:Iron