Xiao Xiansha, Schut Gerrit J, Feng Xiang, Nguyen Diep M N, Huang Haiyan, Wang Shuning, Li Huilin, Adams Michael W W
Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan, USA.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2025 Apr;301(4):108410. doi: 10.1016/j.jbc.2025.108410. Epub 2025 Mar 18.
Flavin-based electron bifurcation couples exergonic and endergonic redox reactions in one enzyme complex to circumvent thermodynamic barriers and minimize free energy loss. Two unrelated enzymes designated NfnSL and NfnABC catalyze the NADPH-dependent reduction of ferredoxin and NAD. Bifurcation by NfnSL resides with a single FAD but the bifurcation mechanism of NfnABC, which represents the diverse and ubiquitous Bfu enzyme family, is completely different and largely unknown. Using cryo-EM structures of an archaeal NfnABC, we show that its bifurcation site is a flavobicluster consisting of FMN, one [4Fe-4S] and one [2Fe-2S] cluster where zinc atoms replace two additional clusters previously identified in other Bfu enzymes. NADH binds to the flavobicluster site of NfnABC and induces conformational changes that allow ferredoxin to bind between the C-terminal domains of NfnC and NfnB. Site-directed mutational analyses support the proposed mechanism that is likely conserved in all members of the Bfu enzyme family.
基于黄素的电子分叉在一个酶复合物中将放能和吸能氧化还原反应偶联起来，以克服热力学障碍并使自由能损失最小化。两种不相关的酶，即NfnSL和NfnABC，催化依赖NADPH的铁氧化还原蛋白和NAD的还原反应。NfnSL的分叉作用由单个FAD完成，但NfnABC的分叉机制则完全不同且很大程度上未知，NfnABC代表了多样且普遍存在的Bfu酶家族。通过对一种古细菌NfnABC的冷冻电镜结构研究，我们发现其分叉位点是一个由FMN、一个[4Fe-4S]和一个[2Fe-2S]簇组成的黄素簇，其中锌原子取代了先前在其他Bfu酶中鉴定出的另外两个簇。NADH结合到NfnABC的黄素簇位点并诱导构象变化，使得铁氧化还原蛋白能够结合在NfnC和NfnB的C末端结构域之间。定点突变分析支持了所提出的机制，该机制可能在Bfu酶家族的所有成员中保守。
