Stepwise assembly of the active site of [NiFe]-hydrogenase

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H₂ into 2e^- and 2H⁺ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)₂(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN^- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O₂-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)₂(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.