The C-helix in CooA Rolls upon CO Binding to Ferrous Heme*
The C-helix in CooA Rolls upon CO Binding to Ferrous Heme*
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DOI：
10.1074/jbc.m407766200
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发表时间：
2004-11
影响因子：
4.8
通讯作者：
Taku Yamashita;Yohei Hoashi;Y. Tomisugi;Y. Ishikawa;T. Uno
中科院分区：
文献类型：
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作者：
Taku Yamashita;Yohei Hoashi;Y. Tomisugi;Y. Ishikawa;T. Uno
CooA是红红螺旋菌的同源二聚体转录激活剂，每个亚基含有一个血红素。CO与血红素传感器区域的结合激活CooA，促进其通过DNA结合区域与DNA的结合。c -螺旋连接两个结构域并形成亚基之间的界面。为了探究CO活化的本质，将c -螺旋上112-121位的残基替换为Asn或Gln，并通过共振拉曼光谱和CO结合亲和力测量来评估其效果。在L116Q、G117N和L120Q突变体中，CO结合野生型CooA的ν(Fe-CO)拉伸拉曼线向上移动了6 cm-1，明确表明这些残基与结合的CO接近。来自每个亚基的Leu116和Leu120残基与相反亚基的相应残基形成接触，从而实现了非活性亚铁形式的疏水相互作用。因此，在CO结合激活的形式中，两个c -螺旋似乎都将这些残基指向血红素，为结合的CO形成疏水袋。CO的亲和力在L112Q、I115Q、L116Q、G117N、L120Q和T121N突变体中大约高一个数量级，但在A114N突变体中降低。这种变化表明这些残基与亚铁和/或co结合形式的血红素接近，并负责CooA的激活。提出了CO活化CooA的滚滑机制。
CooA is a homodimeric transcriptional activator from Rhodospirillum rubrum containing one heme in each subunit. CO binding to the heme in its sensor domain activates CooA, facilitating the binding to DNA by its DNA-binding domain. The C-helix links the two domains and shapes an interface between the subunits. To probe the nature of CO activation, residues at positions 112–121 on the C-helix were replaced by Asn or Gln and their effects were evaluated by resonance Raman spectroscopy and by the measurements of CO binding affinity. The ν(Fe-CO) stretching Raman line in CO-bound wild-type CooA was up-shifted by 6 cm-1 in the L116Q, G117N, and L120Q mutants, indicating unequivocally that these residues are close to the bound CO. Residues Leu116 and Leu120 from each subunit form contacts with the corresponding residues in the opposite subunit, enabling hydrophobic interactions in the inactive ferrous form. Thus, in the CO-bound activated form, both C-helices appear to roll to direct these residues toward the heme, forming a hydrophobic pocket for the bound CO. The CO affinity is approximately one order of magnitude higher in the L112Q, I115Q, L116Q, G117N, L120Q, and T121N mutants but reduced in A114N mutant. The variation indicates that these residues are close to the heme in the ferrous and/or CO-bound forms and are responsible for CooA activation. A roll-and-slide mechanism is proposed for CO activation of CooA.