Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.
Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.
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DOI：
10.1021/bi301336r
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发表时间：
2013-02-05
期刊：
影响因子：
2.9
通讯作者：
Cramer SP
中科院分区：
文献类型：
--
作者：
Kuchenreuther JM;Guo Y;Wang H;Myers WK;George SJ;Boyke CA;Yoda Y;Alp EE;Zhao J;Britt RD;Swartz JR;Cramer SP
The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe–S clusters that facilitate electron transfer to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H2. Here, we have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe–4S] ferredoxin as well as a model complex resembling the [2Fe]H catalytic domain of the H-cluster. In order to enrich the hydrogenase with 57Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with 56Fe-labeled Fe–S clusters was activated in vitro using 57Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe]H subcluster with 57Fe, which NRVS confirms by detecting 57Fe–CO and 57Fe–CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second 57Fe–S cluster. EPR spectroscopy indicates that this 57Fe-enriched metal center is not the [4Fe– 4S]H subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an 56Fe-enriched [4Fe–4S]H cluster during in vitro maturation, providing unambiguous evidence for stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.
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影响因子：
3.5
作者：
Czech, Ilka;Silakov, Alexey;Happe, Thomas
通讯作者：
Happe, Thomas
影响因子：
3.7
作者：
Kuchenreuther, Jon M.;George, Simon J.;Swartz, James R.
通讯作者：
Swartz, James R.
影响因子：
15
作者：
Hambourger, Michael;Gervaldo, Miguel;Moore, Thomas A.
通讯作者：
Moore, Thomas A.
DOI：
10.1073/pnas.86.13.4932
发表时间：
1989-07-01
影响因子：
11.1
作者：
ADAMS, MWW;ECCLESTON, E;HOWARD, JB
通讯作者：
HOWARD, JB
影响因子：
16.6
作者：
Bruschi, Maurizio;Greco, Claudio;De Gioia, Luca
通讯作者：
De Gioia, Luca