Cover Picture: [FeFe]‐Hydrogenase: Defined Lysate‐Free Maturation Reveals a Key Role for Lipoyl‐H‐Protein in DTMA Ligand Biosynthesis (Angew. Chem. Int. Ed. 22/2022)

Semisynthetic maturation of [FeFe]-hydrogenase using [Fe2(μ-SH)2(CN)2(CO)4]2−: key roles for HydF and GTP

“…In a major recent advance, the unknown lysate components were identified as proteins and substrates of the glycine cleavage system (GCS), enabling development of a lysate-free defined maturation system consisting of the maturases HydE, HydF, and HydG, together with GCS components H-protein, T-protein, serine hydroxymethyltransferase (SHMT), serine, and ammonium. 32 This work demonstrated an essential role for the aminomethyl-lipoyl-H-protein (H met ) in the biosynthesis of the DTMA ligand of the H-cluster, 32 confirming serine as the source of the DTMA carbons (as previously shown) 33 and establishing ammonium as the ultimate source of the DTMA nitrogen (Scheme 1). 32 We have hypothesized that H met interacts with the putative dinuclear product of HydE ([(Fe I ) 2 (m-SH) 2 (CN) 2 (CO) 4 ] 2À or [2Fe] E ) complexed to HydF, installing the DTMA ligand to yield the [2Fe] F ([(Fe I ) 2 (DTMA)(CN) 2 (CO) 4 ] 2À ) subcluster (Scheme 2).…”

“…32 This work demonstrated an essential role for the aminomethyl-lipoyl-H-protein (H met ) in the biosynthesis of the DTMA ligand of the H-cluster, 32 confirming serine as the source of the DTMA carbons (as previously shown) 33 and establishing ammonium as the ultimate source of the DTMA nitrogen (Scheme 1). 32 We have hypothesized that H met interacts with the putative dinuclear product of HydE ([(Fe I ) 2 (m-SH) 2 (CN) 2 (CO) 4 ] 2À or [2Fe] E ) complexed to HydF, installing the DTMA ligand to yield the [2Fe] F ([(Fe I ) 2 (DTMA)(CN) 2 (CO) 4 ] 2À ) subcluster (Scheme 2). 32 Here we use biophysical and analytical approaches to demonstrate that [2Fe] E binds to HydF.…”

“…32 We have hypothesized that H met interacts with the putative dinuclear product of HydE ([(Fe I ) 2 (m-SH) 2 (CN) 2 (CO) 4 ] 2À or [2Fe] E ) complexed to HydF, installing the DTMA ligand to yield the [2Fe] F ([(Fe I ) 2 (DTMA)(CN) 2 (CO) 4 ] 2À ) subcluster (Scheme 2). 32 Here we use biophysical and analytical approaches to demonstrate that [2Fe] E binds to HydF. Further, we use hydrogen production assays to establish that the HydF -[2Fe] E complex, combined with our GCS-based defined maturation system (in the absence of HydE and HydG), generates a mature, active [FeFe]-hydrogenase.…”

“…In order to test our hypothesis that H met interacts with [2Fe] E bound to HydF to install the DTMA ligand to yield the [2Fe] F subcluster (Scheme 2), we carried out HydA maturation experiments with HydF, [2Fe] E , HydA, and the H met generation system previously described. 32 This system consists of SHMT, T-protein, serine, and NH 4 Cl, as well as several other small molecule components including GTP (see ESI †). 32 These semisynthetic defined maturation reactions provided [FeFe]hydrogenase activities of 95 mmol min À1 mg À1 (Fig.…”

“…32 This system consists of SHMT, T-protein, serine, and NH 4 Cl, as well as several other small molecule components including GTP (see ESI †). 32 These semisynthetic defined maturation reactions provided [FeFe]hydrogenase activities of 95 mmol min À1 mg À1 (Fig. 3), a number comparable to recent reports for semisynthetic maturation using cell lysate rather than the defined components described here.…”