Smith E T, Adams M W
Department of Biochemistry, University of Georgia, Athens 30602.
Biochim Biophys Acta. 1994 May 18;1206(1):105-12. doi: 10.1016/0167-4838(94)90078-7.
Thermotoga maritima is a H2-producing, fermentative anaerobe and is one of the most thermophilic bacteria known. Its iron-only (Fe-)hydrogenase was previously shown to be a homotetramer and to contain two [4Fe-4S] and two [2Fe-2S] clusters per monomer, but the enzyme lacked the characteristic EPR signal of the oxidized H cluster, the proposed site of H2 catalysis in mesophilic Fe-hydrogenases (Juszczak, A., Aono, S. and Adams, M.W.W. (1991) J. Biol. Chem. 266, 13834-13841). The two types of cluster were shown by spectroelectrochemistry to have reduction potentials (Em) of -390 and -440 mV, respectively. We have now identified two additional redox centers in the enzyme, a [2Fe-2S] center with a higher reduction potential (Em = -365 mV) and an unusual paramagnetic species (Em > -200 mV). The higher potential [2Fe-2S] center can be reduced by sodium dithionite at pH 6.0 and exhibits an axial-type EPR signal with gz = 2.026 and gy = gx = 1.940. The two lower potential [2Fe-2S] centers are fully reduced by sodium dithionite only at pH 10.0. Both of these clusters in their reduced states exhibit rhombic-type EPR signals with gz = 2.005, gy = 1.955, and gx = 1.921. This hydrogenase is therefore thought to contain three [2Fe-2S] clusters, as well as two [4Fe-4S] clusters. In addition, a nearly isotropic EPR signal (g = 2.01) was observed when the enzyme was anaerobically oxidized by organic dyes such as thionine (E alpha = 64 mV) or 2,6-dichlorophenolindophenol (E alpha = 217 mV). This resonance was not observed at 20 K due to relaxation broadening and therefore did not arise from a conventional organic radical. The oxidized enzyme was fully active in an H2 production assay, and also reacted directly with H2. In contrast, the air-oxidized enzyme was inactive and did not exhibit the g = 2.01 EPR signal. This resonance was assigned to a novel paramagnetic species with an approximate Em value of -70 mV. It is thought to be associated with the H2 activating site of this atypical Fe-hydrogenase.
嗜热栖热菌是一种能产生氢气的发酵性厌氧菌，也是已知的最嗜热的细菌之一。其仅含铁（Fe-）的氢化酶先前被证明是一种同四聚体，每个单体含有两个[4Fe-4S]和两个[2Fe-2S]簇，但该酶缺乏氧化态H簇的特征性电子顺磁共振（EPR）信号，而在嗜温Fe-氢化酶中，H簇被认为是H₂催化的位点（Juszczak, A., Aono, S.和Adams, M.W.W. (1991) J. Biol. Chem. 266, 13834 - 13841）。通过光谱电化学表明，这两种类型的簇的还原电位（Em）分别为-390和-440 mV。我们现在已经在该酶中鉴定出另外两个氧化还原中心，一个还原电位较高（Em = -365 mV）的[2Fe-2S]中心和一个不寻常的顺磁物种（Em > -200 mV）。较高电位的[2Fe-2S]中心在pH 6.0时可被连二亚硫酸钠还原，并表现出轴向型EPR信号，gz = 2.026，gy = gx = 1.940。两个较低电位的[2Fe-2S]中心仅在pH 10.0时被连二亚硫酸钠完全还原。这两个簇在还原状态下均表现出菱形型EPR信号，gz = 2.005，gy = 1.955，gx = 1.921。因此，这种氢化酶被认为含有三个[2Fe-2S]簇以及两个[4Fe-4S]簇。此外，当该酶被诸如硫堇（Eα = 64 mV）或2,6-二氯酚靛酚（Eα = 217 mV）等有机染料厌氧氧化时，观察到一个近乎各向同性的EPR信号（g = 2.01）。由于弛豫加宽，在20 K时未观察到该共振，因此它不是由常规有机自由基产生的。氧化态的酶在H₂产生测定中具有完全活性，并且也能直接与H₂反应。相比之下，空气氧化的酶没有活性，并且不表现出g = 2.01的EPR信号。这个共振被归因于一个近似Em值为-70 mV的新型顺磁物种。它被认为与这种非典型Fe-氢化酶的H₂活化位点有关。
