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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• 作者：Jon M. Kuchenreuther ; Yisong Guo ; Hongxin Wang ; William K. Myers ; Simon J. George ; Christine A. Boyke ; Yoshitaka Yoda ; E. Ercan Alp ; Jiyong Zhao ; R. David Britt ; James R. Swartz ; Stephen P. Cramer
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 5, 2013
• 年：2013
• 卷：52
• 期：5
• 页码：818-826
• 全文大小：384K
• 年卷期：v.52,no.5(February 5, 2013)
• ISSN：1520-4995

文摘

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe鈥揝 clusters that facilitate the transfer of an electron 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. 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鈥揝 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鈥揅O and ⁵⁷Fe鈥揅N normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second ⁵⁷Fe鈥揝 cluster. Electron paramagnetic resonance (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 of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.