The final steps of [FeFe]-hydrogenase maturation

• Published in: Proceedings of the National Academy of Sciences - PNAS 2019-08, Vol.116 (32), p.15802-15810
• Main Authors: Lampret, Oliver, Esselborn, Julian, Haas, Rieke, Rutz, Andreas, Booth, Rosalind L., Kertess, Leonie, Wittkamp, Florian, Megarity, Clare F., Armstrong, Fraser A., Winkler, Martin, Happe, Thomas
• Format: Article
• Language: English
• Subjects: Amino acids; Apoproteins - chemistry; Apoproteins - metabolism; Arginine; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biological Sciences; Catalysis; Catalysts; Clostridium - enzymology; Clusters; Electrochemistry; Functional integration; Glycine; Histidine; Holoenzymes - chemistry; Holoenzymes - metabolism; Hydrogen - metabolism; Hydrogenase; Hydrogenase - chemistry; Hydrogenase - metabolism; Iron - metabolism; Ligands; Lysine; Maturation; Models, Molecular; Physical Sciences; PNAS Plus; Residues; Site-directed mutagenesis; Spectroscopy, Fourier Transform Infrared
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1908121116

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.