Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [Fe-57(2)(adt)(CN)(2)(CO)(4)](2-)

Gilbert-Wilson R; Siebel JF; Adamska-Venkatesh A; Pham CC; Reijerse E; Wang HX; Cramer SP; Lubitz W; Rauchfuss TB

Journal of the American Chemical Society, Vol.137, No.28, 8998-9005, 2015

Gilbert-Wilson R, Siebel JF, Adamska-Venkatesh A, Pham CC, Reijerse E, Wang HX, Cramer SP, Lubitz W, Rauchfuss TB

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively Fe-57-labeled binuclear subsite is described. The precursor [Fe-57(2)(adt)(CN)(2)(CO)(4)](2-) was synthesized from the Fe-57 metal, S-8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)(2)[Fe-57(2)(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 Fe-57 enrichment of the [4Fe-4S](H) cluster was realized by reconstitution with (FeCl3)-Fe-57 and Na2S. The H-ox-CO state of [2(57)Fe](H) and [4(57)Fe-4S](H) HydAl was characterized by Mossbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.