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.
Yang
Jianhua
J
Department of Biochemistry, State University of New York, Buffalo, New York 14214, USA.
Ishimori
Koichiro
K
O'Brian
Mark R
MR
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D001665:Binding Sites; D020369:Bradyrhizobium / Q000378:metabolism*; D005294:Ferrochelatase / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C114259:iron response regulator protein, Bacteria; D006418:Heme; D006639:Histidine; D007501:Iron; D005982:Glutathione Transferase; D005294:Ferrochelatase; D000409:Alanine; D010100:Oxygen