Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme
Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme
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DOI：
10.1111/j.1432-1033.1997.00226.x
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发表时间：
1997-02-15
期刊：
影响因子：
--
通讯作者：
Hedderich, R
中科院分区：
文献类型：
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作者：
Kunkel, A;Vaupel, M;Hedderich, R
来自巴氏甲烷八叠球菌甲醇生长细胞的异二硫键还原酶（MbHdrDE）是由46-kDa亚基MbHdrD和23-kDa亚基MbHdrE组成的膜结合酶。该酶已被证明含有0.6摩尔血红素和20摩尔Fe/S每摩尔异二聚体。此外，还检测到了被认为是活性酶的必需组分的亚化学计量量的FAD。我们现在已经获得了完全不含黄素的高产率的活性杂二硫键还原酶的制剂。MbHdrD和MbHdrE编码基因的克隆和测序显示，这两个亚基也缺乏FAD结合基序，它们被发现形成一个转录单位hdrED。因此，MbHdr不同于来自热自养甲烷杆菌的异二硫酰亚胺还原酶（MtHdr），其是由亚基MtHdrA（80 kDa）、MtHdrB（36 kDa）和MtHdrC（21 kDa）组成的黄铁-硫蛋白，亚基HdrA具有黄素结合位点。序列比较显示，MbHdrD的N-末端三分之一，其中包含两个序列基序的[4Fe-4S]簇，是类似于MtHdrC和MbHdrD的C-末端三分之二是类似于MtHdrB。因此，MbHdrD和MtHdrBC是结构上等同的亚基。MbHdrE显示序列相似性的b型细胞色素，在协议的发现，该亚基包含一个血红素。这些和其他结果表明，MbHdrD窝藏的活性位点的杂二硫键还原和黄素不参与催化。由于MbHdrD只含有铁硫簇，二硫键还原涉及一个电子，而不是两个电子转移反应的机制，必须考虑，如操作铁氧还蛋白：硫氧还蛋白还原酶从叶绿体和蓝藻。
Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. in addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrBC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin:thioredoxin reductases from chloroplasts and cyanobacteria.