Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases
Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases
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DOI：
10.1021/bi700659y
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发表时间：
2007-09-04
期刊：
影响因子：
2.9
通讯作者：
Gennis, Robert B.
中科院分区：
文献类型：
--
作者：
Hemp, James;Han, Huazhi;Gennis, Robert B.
血红素-铜超家族的氧还原酶成员是线粒体和许多需氧细菌和古细菌中的末端呼吸氧化酶，将分子氧还原成水与质子跨质膜的易位偶联。催化和泵入氧还原酶所需的质子来自膜的细胞质侧，通过质子传导通道转移，所述质子传导通道由含有内部水分子的氢键链和极性氨基酸侧链组成，所述氢键链沿着。最近的分析确定了超家族中的八个氧还原酶家族：氧还原酶的A-、B-、C-、D-、E-、F-、G-和H-家族。两个质子输入通道，K-通道和D-通道，在氧还原酶的A-家族中得到了很好的建立（例如线粒体细胞色素c氧化酶和来自球形红细菌和嗜热副球菌的呼吸氧化酶）。这些通道中的每一个都可以通过蛋白质内保守的极性氨基酸残基的模式来识别。C家族（cbb（3）氧化酶）是继A家族之后第二丰富的氧还原酶家族，占血红素铜超家族序列的20%以上。在这项工作中，序列分析和结构建模已被用来确定可能的质子通道中的C-家庭。保守极性残基的模式支持仅存在一个质子输入通道，其在空间上类似于A-家族中的K-通道。不存在可以形成D通道类似物或替代质子通道的保守残基模式。建议作为K通道一部分的残基的功能重要性通过使用来自R.类球形菌和霍乱弧菌。几个被认为是K通道的残基在突变后催化活性显著降低：T219 V、Y227 F/Y228 F、N293 D和Y321 F。这些数据强烈表明，在C-家庭只有一个通道的功能，催化和泵送质子的交付。此外，还提出了一对酸性残基，这是完全保守的C-家庭，可能是质子传导出口通道的一部分泵送质子。与这些酸性氨基酸同源的残基在一氧化氮还原酶的cNOR家族中是高度保守的，并且先前已经被认为是质子传导通道的一部分，该质子传导通道将质子从膜的周质侧递送到cNOR家族中的酶活性位点。C家族可能包含一个同源的质子传导通道，该通道将泵送的质子以相反的方向从活性位点传递到周质。
Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb(3) oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb(3) oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.