The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) ₂ (CN)] organometallic precursor

Abstract

The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe–Fe binuclear subcluster: CN−, CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren’t fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO)2(CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO)2(CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13CN-containing carrier affords 13CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a 13C3,15N-Cys–labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1–Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.

Biosynthesis of the catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE, HydF, and HydG

“…hydrogenase has an absolute requirement for HydG for activating hydrogenase activity, 36 replacing HydG and its substrate tyrosine with this proposed HydG product 50 carrier results in high activity, quite comparable to that of the conventional maturation using HydE, HydF, and HydG. 40 Moreover, the resulting EPR signals and 13 C ENDOR (when syn-B is synthetically prepared with 13 C-N) are identical to those of the conventionally maturated H-cluster. This semi-synthesis approach is analogous to the prior work assembling the Hcluster with the pre-synthesized binuclear cluster, 34,35 but now rolled back to the mononuclear Fe level.…”

“…selenocysteine) the cysteine of the synthon was not routinely possible. Here Rao et al 40 show that the cysteine of the synthon is cleaved during the maturation: the sulfur of this cysteine is vectored into the two adt sulfur bridges of the [2Fe] H subunit (as shown by S/Se EXAFS, Fig. 6), while the rest of the cysteine is eliminated (shown by the lack of 13 C and 15 N-pulse EPR signals when using syn-B- 13 C 3 15 Ncysteine and the concomitant appearance of 13 C 3pyruvate in mass spectroscopy).…”

“…An advantage of this cell free synthesis approach is that isotope labels and chemical analogs can be added efficiently as substrates and cofactors in the in vitro reactions, allowing isotope and element sensitive spectroscopies such as pulse EPR and EXAFS to track their incorporation into an isotope or element edited H-cluster. 17,[37][38][39][40][41]…”

Serine is the molecular source of the NH(CH2)2 bridgehead moiety of the in vitro assembled [FeFe] hydrogenase H-cluster

“…[13][14][15][16] More recently, by using a synthetic [(Cys)Fe(CO) 2 (CN)] carrier we have shown that the two sulfur atoms in the adt ligand are derived from the precursor-bound Cys, but that the CH 2 NHCH 2 component is not. 17 Taken together, the biosynthetic origins of the [Fe 2 S 2 (CO) 3 (CN) 2 ] part of the [2Fe] H subcluster are depicted in Scheme 1: all ve diatomic ligands are tailored from tyrosine by HydG; 18 the two sulfur atoms and the two Fe atoms are from the dangler Fe(Cys) site in HydG (which can be reconstituted with Fe 2+ and free Cys in solution 19 ). Remarkably, these components are all delivered to the binuclear cluster assembly in the form of the [(Cys)Fe(CO) 2 (CN)] product of HydG.…”

New Approaches to an Old Problem: Original Techniques in [FeFe]-hydrogenase Research

“…56,57 More recently, this strategy has been utilized to identify possible intermediates in the assembly of the pre-catalyst. [66][67][68] Arguably, the main impact of artificial maturation to-date has been the simplified preparation of the active enzyme 60,69 including site-selective isotopologues, [70][71][72] which also facilitates rapid screening protocols. 30 Another important aspect is the preparation of [FeFe]-hydrogenases in which the diiron site is synthetically modified in order to generate non-natural H-clusters and enzymes with new properties.…”