The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.
Keywords: catalyst; hydrogenase; maturation; organometallic cofactor; redox enzymes.