Nuclear Resonance Vibrational Spectroscopy and Electron Paramagnetic Resonance Spectroscopy of 57Fe-Enriched [FeFe] Hydrogenase Indicate Stepwise Assembly of the H-Cluster

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-duster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H-2. Here, we have used Fe-57 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 Fe-57 nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with Fe-56-labeled Fe-S clusters was activated in vitro using Fe-57-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with Fe-57, which NRVS confirms by detecting Fe-57-CO and Fe-57-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second Fe-57-S cluster. Electron paramagnetic resonance (EPR) spectroscopy indicates that this Fe-57-enriched metal center is not the [4Fe4S](H) subcluster of the H-duster. This finding demonstrates that the CpI hydrogenase retained an Fe-56-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence of stepwise assembly of the H-duster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.