Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR.
In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.
Lee
Jin-Won
JW
Department of Microbiology, Cornell University, Ithaca, New York 14853-8101, USA.
Helmann
John D
JD
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010545:Peroxides / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D004220:Disulfides; D005296:Ferrous Compounds; D010545:Peroxides; D021122:Protein Subunits; D012097:Repressor Proteins; D013451:Sulfonic Acids; D014157:Transcription Factors; C122138:peroxide repressor proteins; D008345:Manganese; D006861:Hydrogen Peroxide; D015032:Zinc