Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima.
Characterization of the Membrane-Associated Electron-Bifurcating Flavoenzyme EtfABCX from the Hyperthermophilic Bacterium Thermotoga maritima.
复制标题
DOI：
10.1021/acs.biochem.3c00473
复制
发表时间：
2023-12-19
期刊：
影响因子：
2.9
通讯作者：
Adams, Michael W. W.
中科院分区：
文献类型：
--
作者：
Ge, Xiaoxuan;Schut, Gerrit J.;Tran, Jessica;Poole II, Farris L.;Niks, Dimitri;Menjivar, Kevin;Hille, Russ;Adams, Michael W. W.
电子分岔是一种能量守恒机制，在这种机制中，单个酶将一个能氧反应与一个能氧反应耦合在一起。异四聚体EtfABCX通过氧化中电位NADH （E°‘ = - 320 mV）来驱动低电位铁氧还蛋白（E°’ = - 450 mV）的还原，同时耦合高电位甲基萘醌（E°' = - 74 mV）的还原。EtfA中的nadh氧化分叉黄嘌呤腺嘌呤二核苷酸（BF-FAD）发生电子分岔，该分岔具有极交叉的半电位，并将第一个高电位电子传递给电子转移FAD，并通过两个铁硫簇最终传递给甲基萘醌。BF-FAD半醌上的低电位电子同时还原铁氧还蛋白。我们在大肠杆菌中表达了编码海洋热热菌EtfABCX的基因，并纯化了EtfABCX全酶和EtfAB亚复合物。利用电子顺磁共振（EPR）跟踪还原铁氧还蛋白的积累，证实了EtfABCX的分岔活性。为了阐明导致分叉能力的结构因素，利用可见光谱和染料连接酶测定监测的EPR和NADH滴定，对紧邻BF-FAD的EtfA中的R38、P239和V242以及EtfB中的R140四个保守残基进行了表征。R38、P239和V242变异表现出减少但仍然显著的分叉活性。尽管仍然被NADH部分还原，R140变体没有分叉活性，电子转移到它的两个[4Fe-4S]簇被阻止。从EtfABCX和其他三个分岔酶家族的分岔机制方面讨论了R140的作用。
Electron bifurcation is an energy-conservation mechanism in which a single enzyme couples an exergonic reaction with an endergonic one. Heterotetrameric EtfABCX drives the reduction of low-potential ferredoxin (E°′ ∼ −450 mV) by oxidation of the midpotential NADH (E°′ = −320 mV) by simultaneously coupling the reaction to reduction of the high-potential menaquinone (E°′ = −74 mV). Electron bifurcation occurs at the NADH-oxidizing bifurcating-flavin adenine dinucleotide (BF-FAD) in EtfA, which has extremely crossed half-potentials and passes the first, high-potential electron to an electron-transferring FAD and via two iron–sulfur clusters eventually to menaquinone. The low-potential electron on the BF-FAD semiquinone simultaneously reduces ferredoxin. We have expressed the genes encodingThermotoga maritimaEtfABCX in E. coli and purified the EtfABCX holoenzyme and the EtfAB subcomplex. The bifurcation activity of EtfABCX was demonstrated by using electron paramagnetic resonance (EPR) to follow accumulation of reduced ferredoxin. To elucidate structural factors that impart the bifurcating ability, EPR and NADH titrations monitored by visible spectroscopy and dye-linked enzyme assays have been employed to characterize four conserved residues, R38, P239, and V242 in EtfA and R140 in EtfB, in the immediate vicinity of the BF-FAD. The R38, P239, and V242 variants showed diminished but still significant bifurcation activity. Despite still being partially reduced by NADH, the R140 variant had no bifurcation activity, and electron transfer to its two [4Fe-4S] clusters was prevented. The role of R140 is discussed in terms of the bifurcation mechanism in EtfABCX and in the other three families of bifurcating enzymes.
登录
查看更多内容
影响因子：
14.8
作者：
Lubner CE;Jennings DP;Mulder DW;Schut GJ;Zadvornyy OA;Hoben JP;Tokmina-Lukaszewska M;Berry L;Nguyen DM;Lipscomb GL;Bothner B;Jones AK;Miller AF;King PW;Adams MWW;Peters JW
通讯作者：
Peters JW
DOI：
10.1073/pnas.2016978118
发表时间：
2021-01-12
影响因子：
11.1
作者：
Feng, Xiang;Schut, Gerrit J.;Adams, Michael W. W.
通讯作者：
Adams, Michael W. W.
影响因子：
5
作者：
Akhtar, M. Kalim;Jones, Patrik R.
通讯作者：
Jones, Patrik R.
影响因子：
4.8
作者：
Duan, H. Diessel;Lubner, Carolyn E.;Miller, Anne-Frances
通讯作者：
Miller, Anne-Frances
影响因子：
5.2
作者：
Buckel W;Thauer RK
通讯作者：
Thauer RK