A Non‐Covalent [4Fe–4S]/[2Fe] Interface in HydF Guides [FeFe]‐Hydrogenase Maturation

Abstract

ABSTRACT Maturation of [FeFe]‐hydrogenase depends on the HydF maturase, a [4Fe–4S]‐containing scaffold protein that assembles and delivers the organometallic [2Fe] subsite of the H‐cluster. Despite extensive research, the function of the HydF [4Fe–4S] cluster and its interaction with the [2Fe] cofactor remains unresolved, with conflicting evidence regarding cyanide linkage isomerism and the functional role of the cubane. Using 57 Fe nuclear resonance vibrational spectroscopy in combination with selective isotopic labeling, we show that the [2Fe] subsite binds adjacent to the [4Fe–4S] cluster without forming a covalent cyanide bridge, while remaining electronically coupled. Complementary protein structure predictions support this arrangement and reconcile prior spectroscopic, mutagenesis, and structural studies. Extending this framework to earlier biosynthetic steps, additional structure predictions suggest that the [4Fe–4S] cluster contributes to assembly of the CH 2 –NH–CH 2 bridge of the [2Fe] site via interactions with the lipoate cofactor of the aminomethyl‐lipoyl H‐protein, thereby positioning reactive components in proximity to the [2Fe] precursor. Together, these results provide a coherent structural and mechanistic framework for HydF function across distinct stages of H‐cluster biosynthesis.