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首页> 外文期刊>Biochemistry >A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced from Studies with Subunit B of Heterodisulfide Reductase from Methanothermobacter marburgensis
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A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced from Studies with Subunit B of Heterodisulfide Reductase from Methanothermobacter marburgensis

机译：富含半胱氨酸的CCG结构域含有从甲烷热杆菌杆菌的亚单二硫化物还原酶的研究中推断出一种新的[4FE-4S]簇结合基序。

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摘要

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]~(3+) cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX_(31-39)CX_(35-36)CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitutio'n. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g_(zyx) = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. ~(57)Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with ~(57)Fe hyperfine couplings very similar to that of CoM-HDR. CoM-~(33)SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S_3(O/N)i geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.
机译：甲状腺二酮酰基甲基硫化物酸还原酶（HDR）与其活性位点（4FE-4S]簇催化甲状腺炎辅酶M（COM-SH）和辅酶B（COB-SH）的异代硫化钠（COM-SS-COB）的可逆减少）。 COM-HDR，在用COM-SH的氧化酶的半反应下产生的基于机械的顺磁性中间体，是一种具有COM-SH作为配体的新型[4FE-4S]〜（3+）簇。甲烷热杆菌的亚基HDRB HDRABC全酶含有两种半胱氨酸的富含序列基序（CX_（31-39）CX_（35-36）CXXC），被称为PFAM数据库中的CCG结构域，并在许多蛋白质中保守。在这里，我们提出了实验证据，即HDRB的C末端CCG结构域绑定了这个不寻常的[4FE-4S]簇。 HDRB在大肠杆菌中产生，随后通过体外重建而插入铁硫簇。在氧化状态下，没有基板的簇表现出菱形EPR信号（G_（ZYX）= 2.015,1.995和1.950），使COM-HDR信号联想。 〜（57）Fe EndoR光谱显示，这种顺磁性物种是[4FE-4S]簇，其〜（57）FE Hyperfine联轴器非常类似于Com-HDR。 COM-〜（33）SH导致EPR信号扩大，并且在添加COM-SH时，簇的中点电位移位到为COM-HDR观察到的值，两者都指示COM-SH对簇的绑定。 HDRB中所有12个半胱氨酸残基的诱变诱变为C末端CCG结构域的四个半胱氨酸作为簇配体。结合先前的含CCG域蛋白质中的COM-HDR样EPR信号的检测，我们的数据表明C末端CCG结构域在该新颖的[4FE-4S]群体的协调中的一般作用。此外，Zn K边缘X射线吸收光谱鉴定了HDRB中的S_3（O / N）几何和HDR全酶的分离的Zn位点。建议N末端CCG结构域为Zn位点提供配体。

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  • 来源
    《Biochemistry》 |2007年第44期|共11页
  • 作者

    Nils Hamann; Gerd J. Mander; Jacob E. Shokes;

  • 作者单位

    Max Planck Institute for Terrestrial Microbiology Karl-von-Frisch Strasse D-35043 Marburg Germany;

    Department of Chemistry and Center for Metalloenzyme Studies University of Georgia Athens Georgia 30602-2556;

    Institute of Physical and Theoretic;

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  • 正文语种 eng
  • 中图分类 生物化学;
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