Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.
Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.
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通过亚基内交联探测大肠杆菌复合物 I 亚基 N 中的质子通道。
DOI：
10.1016/j.bbabio.2016.09.005
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Vik,StevenB
中科院分区：
文献类型：
--
作者：
Tursun,Ablat;Zhu,Shaotong;Vik,StevenB
呼吸复合体I有4个质子转运点，分别与NADH的氧化和辅酶Q的还原有关。质子通道被认为是由连接到膜表面的偏置的半通道组成，并通过一条穿过膜中心的水平通道连接。在对大肠杆菌中酶的研究中，N亚基是包含其中一个位点的靶标。半胱氨酸残基对沿着所提出的质子路径被引入相邻的α-螺旋中。为了限制在质子转移过程中可能发生的构象变化，我们试图在两个工程半胱氨酸残基之间形成二硫键或甲硫磺酸桥。半胱氨酸的修饰是由于聚乙二醇马来酰亚胺不能改变N亚基的电泳迁移率，而N亚基与游离的巯基反应时会发生这种变化。交联化处理后，检测NADH氧化酶和NADH驱动的质子转运。十对不同的半胱氨酸残基显示出交联性的证据。酶活性的最大损失出现在基本赖氨酸395附近的残留物中。这种残基位于所提出的质子到周质的半通道和通过N亚基的水平连接之间，也位于M亚基的基本Glu 144附近。结果表明，对于将质子输送到周质或将N亚基的作用耦合到M亚基，该区域发生了重要的构象变化。
Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme fromEscherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.