Lee Jin-Won, Helmann John D
Department of Microbiology, Cornell University, Ithaca, New York 14853-8101, USA.
J Biol Chem. 2006 Aug 18;281(33):23567-78. doi: 10.1074/jbc.M603968200. Epub 2006 Jun 8.
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+.
在枯草芽孢杆菌中，大多数过氧化物诱导的氧化应激基因受金属依赖性阻遏物PerR调控。PerR是一种含锌二聚体金属蛋白，具有一个结合Fe2+（PerR:Zn,Fe）或Mn2+（PerR:Zn,Mn）的调节性金属结合位点。PerR:Zn,Fe与低水平过氧化氢（H2O2）反应会导致两个组氨酸残基氧化，从而导致去阻遏。当与Mn2+结合时，形成的PerR:Zn,Mn对氧化失活的敏感性要低得多。在此我们证明，结构锌离子（Zn2+）在一个高度稳定的亚基内Cys4:Zn2+位点配位。H2O2对该Cys4:Zn2+位点的氧化导致亚基内二硫键的形成。氧化速率过慢，无法解释微摩尔水平的H2O2诱导的过氧化物应激反应，但可能在严重氧化应激条件下的诱导过程中起作用。体内研究表明，PerR:Zn,Mn的失活需要10 mM H2O2，这一水平比PerR:Zn,Fe失活所需水平至少高1000倍。令人惊讶的是，即使在这些严重氧化条件下，体内半胱氨酸残基几乎没有可检测到的氧化：去阻遏与调节位点的氧化相关。因为在体外该位点的氧化需要结合的Fe2+，我们认为用10 mM H2O2处理细胞会使足够的Fe2+释放到细胞质中，从而使PerR从PerR:Zn,Mn形式转变为对过氧化物敏感的PerR:Zn,Fe形式。金属离子亲和力测量结果支持了这一模型，表明PerR对Fe2+的亲和力高于Mn2+。
