Abstract
The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively (57)Fe-labeled binuclear subsite is described. The precursor [(57)Fe2(adt)(CN)2(CO)4](2-) was synthesized from the (57)Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[(57)Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary (57)Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with (57)FeCl3 and Na2S. The Hox-CO state of [2(57)Fe]H and [4(57)Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Carbon Monoxide / metabolism
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Catalytic Domain
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Chlamydomonas reinhardtii / chemistry
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Chlamydomonas reinhardtii / enzymology*
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Chlamydomonas reinhardtii / metabolism
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Electron Spin Resonance Spectroscopy*
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Hydrogenase / antagonists & inhibitors
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Hydrogenase / chemistry*
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Hydrogenase / metabolism
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Iron Compounds / chemistry*
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Iron Isotopes / chemistry
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Iron-Sulfur Proteins / antagonists & inhibitors
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Iron-Sulfur Proteins / chemistry*
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Iron-Sulfur Proteins / metabolism
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Models, Molecular
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Spectroscopy, Mossbauer*
Substances
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Iron Compounds
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Iron Isotopes
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Iron-Sulfur Proteins
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Carbon Monoxide
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iron hydrogenase
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Hydrogenase