H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle
H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle
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DOI：
10.1016/j.bbagen.2017.10.002
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发表时间：
2018-01-01
影响因子：
3
通讯作者：
Bothner, Brian
中科院分区：
文献类型：
--
作者：
Berry, Luke;Poudel, Saroj;Bothner, Brian
铁氧还蛋白依赖性转氢酶是一类负责电子传递的酶，最近的研究强调了黄素基电子分叉（FBEB）的生物学重要性。FBEB通过将具有中等电位的电子供体的氧化与高电位和低电位分子的还原偶联来产生具有非常低的还原电位的生物分子。分叉系统可以从诸如NADPH的物质产生具有非常低的还原电位的生物分子，诸如还原的铁氧还蛋白（Fd）。使用分叉的代谢系统效率更高，并为拥有它们的生物体提供竞争优势。现在有两种NADH依赖性铁氧还蛋白-NADP氧化还原酶（Nfn）复合物的结构模型。这些模型，连同光谱研究，提供了相当深入的了解FBEB的催化过程。然而，关于这些多亚基蛋白的机制和调控仍不清楚。使用氢/氘交换质谱（HDX-MS）和统计耦合分析（SCA），我们确定了特定的通信途径内的模型FBEB系统，Nfn从Pyrococus firiosus，在催化循环的每一步的条件下。HDX-MS揭示了在核苷酸和铁氧还蛋白结合后跨蛋白质亚基的变构偶联的证据。SCA发现了一个共同进化的残基网络，可以在整个复合体中提供连接。总之，HDX-MS和SCA数据表明，蛋白质变构发生在铁硫辅因子和配体结合位点的集合中，使用连接结构域的特定途径，使它们作为动态协调单元发挥作用。
Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus fiiriosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron-sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.