Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement
Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement
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DOI：
10.1111/1462-2920.13200
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发表时间：
2016-09-01
影响因子：
5.1
通讯作者：
Adrian, Lorenz
中科院分区：
文献类型：
--
作者：
Kublik, Anja;Deobald, Darja;Adrian, Lorenz
Mccartyi Dehalococcoides菌株CBDB 1是一株仅以氢为电子供体、卤代有机物为电子受体的专性有机卤化物呼吸细菌。在这里，我们研究了在非变性条件下参与呼吸链的蛋白质。采用蓝色非变性凝胶电泳（BN-PAGE）、凝胶过滤和超滤技术，鉴定出一种分子量为250-270 kDa的活性脱卤蛋白复合物。还原性脱卤酶（RdhA）的活性亚基与铁硫复合脱卤酶（CISM）亚基（CbdbA 195）和氢吸收氢化酶（Hup）的含铁硫簇的亚基（CbdbA 131）共定位。氢化酶和还原性脱卤酶的催化活性亚基之间没有共定位被发现。通过二维BN/SDS-PAGE对洗涤剂的复合物的稳定性进行了评估，表现出逐步解体随着洗涤剂浓度的增加。化学交联证实了由RdhA、CISM I和Hup氢化酶组成的更高分子量还原性脱卤酶蛋白复合物的存在，并被证明是在膜溶解之前稳定脱卤复合物的蛋白质-蛋白质相互作用的潜在工具。总之，呼吸脱卤酶蛋白复合物的鉴定和醌参与呼吸的迹象的缺乏表明，在D.麦卡蒂。
Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under nondenaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.