The [4Fe-4S] Cluster of HydF Is Essential for [FeFe]-Hydrogenase Maturation

The organometallic H-cluster of the [FeFe]-hydrogenase is assembled in vivo through a complex process requiring the action of three dedicated maturation enzymes, HydG, HydE, and HydF, as well as the aminomethyl-lipoyl-H-protein (H_met) of the glycine cleavage system (GCS). Here we probe the role of HydF and its [4Fe-4S] cluster in [FeFe]-hydrogenase maturation by using a defined semisynthetic approach in which [Fe^I₂(μ-SH)₂(CO)₄(CN)₂]^2- ([2Fe]_E) is used to bypass HydE and HydG, and GCS components are used in place of cell lysate. We show that inclusion of the iron-sulfur carrier protein NfuA and the high-CO-affinity myoglobin variant Mb^H64L provides dramatically improved hydrogenase activities up to 828 μmol/min/mg, equivalent to the best reported activities for Chlamydomonas reinhardtii [FeFe]-hydrogenase isolated from the native organism. Apo-HydF lacking a [4Fe-4S] cluster provides very little hydrogenase activity; however, full maturation is restored with the addition of NfuA, which we demonstrate reconstitutes the [4Fe-4S] cluster of HydF. In addition, a HydF variant lacking a [4Fe-4S] cluster by changing two cysteine ligands to alanine is completely unable to support either semisynthetic maturation using [2Fe]_E, or full maturation using HydG and HydE, even in the presence of NfuA, demonstrating that the HydF [4Fe-4S] cluster is absolutely essential for [FeFe]-hydrogenase maturation. The possibility that the HydF [4Fe-4S] cluster plays a role in direct binding of [2Fe]_E is negated by our results with the HydF^D311C variant, which demonstrate that the labile Asp311 cluster ligand is not essential for [2Fe]_E binding and HydA maturation. We therefore conclude that [2Fe]_E binds HydF adjacent to, but not directly coordinated to, the [4Fe-4S] cluster. The HydF [4Fe-4S] cluster is proposed to be essential due to its impact on the [2Fe]_E binding orientation and the ability of the HydF/[2Fe]_E complex to form productive interactions with H_met or the H_met/T-protein complex during DTMA ligand biosynthesis.