Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy
Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy
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DOI：
10.1042/bj20070669
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发表时间：
2007-11-15
影响因子：
4.1
通讯作者：
Butler, Clive S.
中科院分区：
文献类型：
--
作者：
Dridge, Elizabeth J.;Watts, Carys A.;Butler, Clive S.
来自Thauera selenatis的周质SER（硒酸盐还原酶）被分类为达特（双精氨酸移位酶）移位（II型）腺苷酸酶的成员，并且包括三个亚基，每个亚基含有氧化还原辅因子。变温X-带EPR谱显示了[3Fe-4S]（1+）、[4Fe-4S]（1+）、Mo（V）和血红素中心的特征。EPR监测的SerABC复合物（SerA-SerB-SerC，任何亚基的异三重复合物）的氧化还原电位滴定显示，[3Fe-4S]簇（FS 4，铁-硫簇4）作为n=1能斯特组分滴定，其中[3Fe-4S]（1+/0）对的中点氧化还原电位（E1）为+118 +/- 10 mV。[4Fe-4S]（1+）团簇EPR信号在300和- 200 mV之间的电位范围内产生，并且最好地拟合两个连续的能斯特n= 1曲线，其中两个[4Fe-4S]（1+/2+）团簇对的中点氧化还原电位为+ 183 +/-10 mV（FS 1）和-51 +/- 10 mV（FS 3）。进一步还原后，[4Fe-4S]（1+）团簇的信号强度降低。这种强度的变化可以再次拟合到具有约-356 mV（FS 2）的中点电位（Em）的n= 1能斯特分量。据认为，在低氧化还原电位（Em小于-300 mV），剩余的氧化簇被还原（自旋S =1/2），并强烈自旋耦合到相邻的[4Fe 4S]（1+）簇，使两个中心EPR沉默。参与的[3Fe-4S]和[4Fe-4S]集群的电子转移到周质SER的活性位点的再氧化的集群下厌氧硒酸营业额条件下证明。在低温（5 K）和高功率（100 mW）条件下探测SerA中的高自旋[4Fe-4S]团簇（FS 0）的尝试没有成功。在pH 6.0的样品中，在60 K下记录的Mo（V）EPR显示g（3）类似于1.999，g（2）类似于1.996和g（1）类似于1.965（g（av）1.9867）的主g值。在g（2）和g（3）处的主要特征没有分裂，但在光谱的g（1）区域观察到超精细分裂，并且可以最好地模拟为由耦合常数为A（1）（H-1）=1.014 mT的单个质子产生。在SerC中的血红素-b部分的存在下，通过检测到类似于3.33的g处的信号来证明，并且与由甲硫氨酸和赖氨酸轴向配体配位的血红素一致。结合EPR分析和序列比对的证据支持分配的周质SER作为11型辅酶酶的成员，并提供了第一个光谱电位洞察酶催化的关键还原反应的硒地球化学循环。
Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S](1+), [4Fe-4S](1+), Mo(V) and haemh. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of any subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E,) of +118 +/- 10 mV for the [3Fe-4S](1+/0) couple. A [4Fe-4S](1+) cluster EPR signal developed over a range of potentials between 300 and - 200 mV and was best fitted to two sequential Nernstian n= 1 curves with midpoint redox potentials of + 183 +/- 10 mV (FS 1) and -51 +/- 10 mV (FS3) for the two [4Fe-4S](1+/2+) cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S](1+) cluster decreases. This change in intensity can again be fitted to an n= 1 Nernstian component with a midpoint potential (Em) of about -356 mV (FS2). It is considered likely that, at low redox potential (Em less than -300 mV), the remaining oxidized cluster is reduced (spin S =1/2) and strongly spin-couples to a neighbouring [4Fe4S](1+) cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g(3) similar to 1.999, g(2) similar to 1.996 and g(1) similar to 1.965 (g(av) 1.9867). The dominant features at g(2) and g(3) are not split, but hyperfine splitting is observed in the g(1) region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A(1) (H-1) =1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g similar to 3.33 and is consistent with haem coordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type 11 molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.