Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein
Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein
复制标题
DOI：
10.1074/jbc.ra120.013174
复制
发表时间：
2020-09-04
影响因子：
4.8
通讯作者：
Miller, Anne-Frances
中科院分区：
文献类型：
--
作者：
Duan, H. Diessel;Mohamed-Raseek, Nishya;Miller, Anne-Frances
在沼泽红球藻的分叉电子转移黄素蛋白（Bf-ETF）（RpaETF）中发现了一个显著的电荷转移（CT）带，RpaETF含有两个FAD，它们在电子分叉中起着不同的作用。Bf-FAD从NADH成对接受电子，将一个引导到较低还原中点电位（E度）载体，而另一个引导到较高E度电子转移FAD（ET-FAD）。先前的工作指出，当ET-FAD被还原而Bf-FAD被氧化时，在726 nm处形成CT带，这表明两种黄素都参与其中。然而，现有的晶体结构使它们相距太远，无法直接相互作用。我们目前的生化实验解决这个难题，并阐明这种CT物种的性质。我们观察到缺少FAD的RpaETF缺少726 nm波段。定点突变附近的任一FAD产生改变产量的CT物种，支持参与这两个黄素。残基取代没有改变信号的最大吸收，排除了残基轨道的贡献。相反，我们建议，残基身份调节人口的蛋白质构象，使ET-黄素和BF-黄素直接接触，解释了726 nm波段的基础上CT复合物的减少ET-FAD和氧化BF-FAD。这是证实了持久性的726 nm物种在温和的蛋白质变性和简单的密度泛函理论计算黄素二聚体。虽然这种CT复合物已被证明为游离黄素，这是第一次观察到这样的，据我们所知，在酶。因此，Bf-ETF可以通过实现直接黄素-黄素接触来优化电子转移效率。
A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) fromRhodopseudomonas palustris(RpaETF).RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E degrees) carrier, and the other to the higher-E degrees electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed thatRpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.