The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803
The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803
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DOI：
10.1074/jbc.ra120.013136
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发表时间：
2020-07-10
影响因子：
4.8
通讯作者：
King, Paul W.
中科院分区：
文献类型：
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作者：
Artz, Jacob H.;Tokmina-Lukaszewska, Monika;King, Paul W.
蓝藻HOX是一种由氢(H-2)激活亚基HoxYH组成的[NiFe]氢酶，HoxYH与HoxEFU组装形成复合体，介导与可溶性电子载体NAD(P)H和铁氧还蛋白(FDX)的反应，从而将光合作用电子传递耦合到能量转换催化反应中。研究HoxEFUYH复合体的研究人员观察到，HoxEFU可以独立于HoxYH分离出来，这导致了HoxEFU是一个独特的功能亚复合体的假说，而不是HOX复合体分离的产物。此外，HOX与天然底物的反应活性以及底物相互作用和偶联H-2、NAD(P)H和FDX的位置(S)仍有待解决。为了解决这些问题，我们用天然底物对重组产生的HoxEFU进行了电子顺磁共振波谱和动力学分析。纯化的HoxEFU亚复合体催化NAD(P)H、黄还蛋白和几种铁氧还蛋白之间的电子转移反应，从而在体外起到在不同还原当量蓝藻之间穿梭的作用。依赖Fdx1的NAD(+)和NADP(+)的减少都是协同的。HoxEFU还催化黄毒素依赖的NAD(P)(+)还原，Fdx2依赖的NADH氧化，Fdx4-和Fdx11依赖的NAD(+)还原。基于MS的图谱在HoxE和HoxF的交界处发现了一个Fdx1结合位点，与两个亚基中的铁硫(FeS)簇相邻。总体而言，这里观察到的HoxEFU的反应性表明，它在管理来自光合作用电子转移的外周电子流方面发挥作用，这一发现揭示了无处不在的细胞成分如何被用来将能量流分配到特定的生物能量产品的详细见解。
Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H-2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H-2, NAD(P)H, and Fdx remain to be resolved. To address these questions, here we analyzed recombinantly produced HoxEFU by electron paramagnetic resonance spectroscopy and kinetic assays with natural substrates. The purified HoxEFU subcomplex catalyzed electron transfer reactions among NAD(P)H, flavodoxin, and several ferredoxins, thus functioningin vitroas a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD(+)and NADP(+)were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)(+), Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD(+). MS-based mapping identified an Fdx1-binding site at the junction of HoxE and HoxF, adjacent to iron-sulfur (FeS) clusters in both subunits. Overall, the reactivity of HoxEFU observed here suggests that it functions in managing peripheral electron flow from photosynthetic electron transfer, findings that reveal detailed insights into how ubiquitous cellular components may be used to allocate energy flow into specific bioenergetic products.