Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli
Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli
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DOI：
10.1128/jb.02335-14
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发表时间：
2015-01-01
影响因子：
3.2
通讯作者：
Sawers, R. Gary
中科院分区：
文献类型：
--
作者：
Pinske, Constanze;Jaroschinsky, Monique;Sawers, R. Gary
大肠杆菌摄取氢化酶2（Hyd2）催化H-2可逆氧化为质子和电子。在甘油或甘油-富马酸盐上生长期间，Hyd-2合成强烈上调。膜相关Hyd-2是一种不寻常的异源四聚体[NiFe]-氢化酶，其缺乏典型的细胞色素B膜锚亚基，该亚基将电子转移到醌池。相反，Hyd-2有一个额外的电子转移亚基，称为HybA，有四个预测的铁硫簇。在这里，我们研究了HybA亚基的生理作用。在甘油和富马酸盐的呼吸生长过程中，Hyd-2使用甲萘醌/去甲基甲萘醌（MQ/DMQ）将氢氧化与富马酸盐还原偶联。HybA是Hyd-2向MQ/DMQ的电子转移所必需的。在酪蛋白氨基酸存在下或在与甘油-富马酸盐一起生长的富马酸盐还原酶阴性菌株中在甘油发酵期间由Hyd-2催化的H-2释放也显示依赖于HybA和MQ/DMQ两者。解偶联剂羰基氰化物间氯苯腙（CCCP）抑制Hyd2-依赖性的H-2从甘油中释放，表明需要质子梯度。相比之下，CCCP未能抑制H-2偶联富马酸盐减少。虽然缺乏HybA的Hyd-2酶不能催化全细胞中的Hyd-2依赖性H-2氧化或H-2进化，但仍然发生紫精染料的可逆H-2依赖性还原。最后，氢依赖性染料还原Hyd-2可逆地抑制提取物来自H-2进化模式生长的细胞。我们的研究结果表明，Hyd-2开关之间的H-2-消耗和H-2-生产模式响应醌池的氧化还原状态。Hyd2依赖性的从甘油的H-2进化需要反向电子传递。
Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H-2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H-2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H-2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H-2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H-2 oxidation or H-2 evolution in whole cells, reversible H-2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H-2 evolution mode. Our findings suggest that Hyd-2 switches between H-2-consuming and H-2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H-2 evolution from glycerol requires reverse electron transport.