Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans
Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans
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DOI：
10.1074/jbc.m114.617761
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发表时间：
2015-01-09
影响因子：
4.8
通讯作者：
Jahn, Dieter
中科院分区：
文献类型：
--
作者：
Kiesel, Svenja;Waetzlich, Denise;Jahn, Dieter
细菌叶绿素a的生物合成需要叶绿素a的C7-C8双键被固氮酶样多亚基金属酶叶绿素a氧化还原酶（COR）进行立体和区域特异性的双电子还原。ATP依赖性COR催化需要蛋白质亚复合物（BchX）（2）与催化蛋白质（BchY/BchZ）（2）的相互作用，以通过两个氧化还原活性铁-硫中心促进底物还原。在ATP过渡态类似物ADP. AlF 4-的存在下，捕获来自紫色细菌Roseobacterium pesticans的包含亚基BchX、BchY和BchZ的三元COR酶全复合物。电子顺磁共振实验表明，[4Fe-4S]簇的子配合物（BchX）（2）。第二个[4Fe-4S]簇在（BchY/BchZ）（2）上鉴定。诱变实验表明，后者是由四个半胱氨酸连接，这是在相反的密切相关的darkoperative原叶绿素a氧化还原酶（DPOR）的三个半胱氨酸/一个天冬氨酸连接模式。在随后的诱变实验中，对COR的催化[4Fe-4S]簇实施DPOR样天冬氨酸连接模式。光谱证明了这种无活性COR变体的人工簇形成。一系列的化学改性的底物分子与改变取代基的个别吡咯环和isocyclic环进行了测试作为COR底物。COR酶仍然能够还原在环系统A、C和E上携带修饰取代基的底物的B环。然而，基板与位于远处的丙酸侧链的修改不被接受。与相关的DPOR系统类似，得出了一个初步的底物结合模式。
Bacteriochlorophyll a biosynthesis requires the stereo-and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)(2) with the catalytic (BchY/BchZ)(2) protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP.AlF4-. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)(2). Asecond [4Fe-4S] cluster was identified on (BchY/BchZ)(2). Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/ one aspartate ligation pattern of the closely related darkoperative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.