Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation
| Autor: | Robert J. Usselman, Joan B. Broderick, Krista A. Shisler, Eric M. Shepard, Jeremiah N. Betz, Priyanka Aggarwal, Gareth R. Eaton, Amanda S. Byer, Anna G. Scott, Sandra S. Eaton |
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| Rok vydání: | 2017 |
| Předmět: |
Iron-Sulfur Proteins
0301 basic medicine S-Adenosylmethionine Hydrogenase Protein Conformation Stereochemistry Iron Cluster chemistry GTPase 010402 general chemistry 01 natural sciences Biochemistry Catalysis Article law.invention 03 medical and health sciences chemistry.chemical_compound Biosynthesis law Catalytic Domain Organic chemistry Electron paramagnetic resonance Clostridium chemistry.chemical_classification Chemistry Electron Spin Resonance Spectroscopy 0104 chemical sciences 030104 developmental biology Enzyme Oxidation-Reduction Sulfur Cysteine |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.7b00169 |
| Popis: | Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway. |
| Databáze: | OpenAIRE |
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