Artificially maturated [FeFe] hydrogenase from Chlamydomonas reinhardtii: a HYSCORE and ENDOR study of a non-natural H-cluster

作者：Adamska Venkatesh Agnieszka; Simmons Trevor R; Siebel Judith F; Artero Vincent; Fontecave Marc; Reijerse Edward^*; Lubitz Wolfgang

来源：Physical Chemistry Chemical Physics, 2015, 17(7): 5421-5430.

DOI：10.1039/c4cp05426a

Hydrogenases are enzymes that catalyze the oxidation of H-2 as well as the reduction of protons to form H-2. The active site of [FeFe] hydrogenase is referred to as the "H-cluster'' and consists of a "classical'' [4Fe-4S] cluster connected via a bridging cysteine thiol group to a unique [2Fe](H) sub-cluster, containing CN- and CO ligands as well as a bidentate azadithiolate ligand. It has been recently shown that the biomimetic [Fe-2(adt)(CO)(4)(CN)(2)](2-) (adt(2-) = azadithiolate) complex resembling the diiron sub-cluster can be inserted in vitro into the apo-protein of [FeFe] hydrogenase, which contains only the [4Fe-4S] part of the H-cluster, resulting in a fully active enzyme. This synthetic tool allows convenient incorporation of a variety of diiron mimics, thus generating hydrogenases with artificial active sites. [FeFe] hydrogenase from Chlamydomonas reinhardtii maturated with the biomimetic complex [Fe-2(pdt)(CO)(4)(CN)(2)](2-) (pdt(2-) = propanedithiolate), in which the bridging adt(2-) ligand is replaced by pdt(2-), can be stabilized in a state strongly resembling the active oxidized (H-ox) state of the native protein. This state is EPR active and the signal originates from the mixed valence (FeFeII)-Fe-I state of the diiron sub-cluster. Taking advantage of the variant with N-15 and C-13 isotope labeled CN- ligands we performed HYSCORE and ENDOR studies on this hybrid protein. The C-13 hyperfine couplings originating from both CN- ligands were determined and assigned. Only the N-15 coupling from the CN- ligand bound to the terminal iron was observed. Detailed orientation selective ENDOR and HYSCORE experiments at multiple field positions enabled the extraction of accurate data for the relative orientations of the nitrogen and carbon hyperfine tensors. These data are consistent with the crystal structure assuming a g-tensor orientation following the local symmetry of the binuclear sub-cluster.

• 出版日期2015
• 单位中国地震局