The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies.
The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies.
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来自 Desulfovibrio gigas 的腺苷酸硫酸还原酶的活性中心。
DOI：
10.1111/j.1432-1033.1990.tb15447.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Moura,JJ
中科院分区：
文献类型：
--
作者：
Lampreia,J;Moura,I;Teixeira,M;PeckJr,HD;Legall,J;Huynh,BH;Moura,JJ
为了利用硫酸盐作为末端电子受体，硫酸盐还原菌配备了一个复杂的酶系统，其中腺苷酰硫酸盐（ADAPSO 4）还原酶发挥主要作用之一，将ADAPSO 4（硫酸盐的活化形式）还原为亚硫酸盐，并释放AMP。从厌氧硫酸盐还原菌Desulfovibrio gigas中分离纯化得到了均一的硫酸盐还原酶。该蛋白由两个不同的亚基（70 kDa和23 kDa）组成，并以多聚体形式（约400 kDa）分离。它是一种含铁硫、黄素的蛋白质，具有一个FAD部分、八个铁原子和93 kDa的最小分子量。在天然状态下，该酶显示出以g = 2.02为中心的几乎各向同性的信号，并且仅在20 K以下可检测到。该信号代表一种次要物质（0.10-0.25 spins/mol），并显示从57 Fe生长细胞中分离的酶中的谱线增宽。添加亚硫酸盐对EPR光谱的影响较小，但导致光谱的可见光区（约392 nm）的主要降低。进一步添加AMP仅引起可见光谱的微小变化，而在EPR光谱中观察到主要变化;在低于30 K的温度下观察到g值为2.096、1.940和1.890的菱形信号（还原的Fe-S中心I）的出现，以及g = 2.02信号强度的伴随降低。还研究了化学还原剂（抗坏血酸盐、H2/氢化酶还原的甲基紫精和连二亚硫酸盐）的影响。用连二亚硫酸盐短时间还原（15 s）或用甲基紫精还原，使中心I完全还原（g值在2.079、1.939和1.897处略有改变），g = 2.02信号完全消失。用连二亚硫酸盐进一步还原产生非常复杂的自旋-自旋耦合性质的EPR谱（在20 K以下可观察到），表明至少存在两个铁硫中心（中心I和II）。gigasophosphorus PSO 4还原酶明确地证明了两个4Fe簇的存在。中心II具有400 mV的氧化还原电位，并表现出铁氧还蛋白-型[4Fe-4S]簇的光谱特性。中心I在其还原态下表现出具有非典型穆斯堡尔参数的光谱，并且具有约0 mV的中点电位，这与铁氧还蛋白型[4Fe-4S]簇的中点电位不同，表明不同的结构和/或不同的簇配体环境。
In order to utilize sulfate as the terminal electron acceptor, sulfate‐reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4(the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducerDesulfovibrio gigas. The protein is composed of two non‐identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (∼ 400 kDa). It is an iron‐sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa.Low‐temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered atg= 2.02 and only detectable below 20 K. This signal represented a minor species (0.10–0.25 spins/mol) and showed line broadening in the enzyme isolated from57Fe‐grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal atgvalues 2.096, 1.940 and 1.890 (reduced Fe‐S center I) observable below 30 K and a concomitant decrease in intensity of theg= 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase‐reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modifiedgvalues at 2.079, 1.939 and 1.897), and the complete disappearance of theg= 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin–spin‐coupled nature (observable below 20 K), indicating the presence of at least two iron‐sulfur centers, (centers I and II).Mössbauer studies on57Fe‐enrichedD. gigasAdoPSO4reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin‐type [4Fe‐4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin‐type [4Fe‐4S] cluster, suggesting a different structure and/or a distinct cluster‐ligand environment.
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DOI：
10.1016/s0926-6593(65)80141-2
发表时间：
1965
期刊：
Biochimica et Biophysica Acta
影响因子：
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作者：
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DOI：
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发表时间：
1978
期刊：
Biochimica et Biophysica Acta
影响因子：
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作者：
Isabel Moura;António V. Xavier;Richard Cammack;M. Bruschi;J. Gall
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影响因子：
15
作者：
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影响因子：
2.7
作者：
M. Ishimoto;D. Fujimoto
通讯作者：
D. Fujimoto
来自巴氏梭菌的 2[4Fe-4Se]+ 铁氧还蛋白的 EPR 和穆斯堡尔谱中的异常特征。
DOI：
10.1016/s0021-9258(18)90874-4
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
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作者：
J. Moulis;P. Auric;J. Gaillard;J. Meyer
通讯作者：
J. Meyer