Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris
Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris
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DOI：
10.1074/jbc.ra117.000707
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发表时间：
2018-03-30
影响因子：
4.8
通讯作者：
Miller, Anne-Frances
中科院分区：
文献类型：
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作者：
Duan, H. Diessel;Lubner, Carolyn E.;Miller, Anne-Frances
电子分叉是生物学中能量守恒的第三种基本机制，它利用放能氧化还原反应的自由能来驱动吸能氧化还原反应。基于黄素的电子分叉将低电位电子转移到要求苛刻的化学反应中，例如将二氮还原为氨。我们采用异源二聚体黄素酶FixAB从固氮细菌Rhodopyramidpalustris阐明独特的属性，支持黄素为基础的电子分叉。FixAB与典型电子转移黄素蛋白（ETF）的区别在于第二个FAD，它取代了典型ETF的AMP。我们利用近紫外-可见CD光谱来解析来自FixAB中不同黄素位点的信号，并询问假定的分叉FAD。CD有助于将测得的还原中点电位（E度值）分配给单个黄素，E度值测试了有关分叉所需氧化还原性质的公认模型。我们发现，较高的E度黄素显示连续的单电子（1-e（-））还原为阴离子半醌，然后对苯二酚，与典型的ETF中看到的反应性一致。与此相反，较低的E度黄素显示一个单一的两电子（2-e（-））减少没有检测到的半醌积累，与不稳定的半醌状态，所需的分叉。这是第一次证明FixAB蛋白具有分叉活性的热力学先决条件，并且两种黄素的不同光学特征的分离为机械研究奠定了基础，以了解如何在蛋白质环境中引导电子流。我们建议，在长波长观察到的一种新的光信号可能反映了电子离域两个黄素之间。
A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E degrees values) to individual flavins, and the E degrees values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E degrees flavin displays sequential one-electron (1-e(-)) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E degrees flavin displayed a single two-electron (2-e(-)) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.