9RKM

Crystal structure of [FeFe]-hydrogenase CbA5H from Clostridium beijerinckii in Hinact state

Summary for 9RKM

• Entry DOI: 10.2210/pdb9rkm/pdb
• Descriptor: [FeFe]-hydrogenase, dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+), IRON/SULFUR CLUSTER, ... (9 entities in total)
• Functional Keywords: fefe hydrogenase hydrogen oxygen stability, oxidoreductase
• Biological source: Clostridium beijerinckii
• Total number of polymer chains: 2
• Total formula weight: 147983.75

Authors

Carr, S.B.,Alogaidi, A.,Morra, S. (deposition date: 2025-06-13, release date: 2026-06-24, Last modification date: 2026-08-05)

Primary citation

Alogaidi, A.,Carr, S.B.,Hudson, L.,Lloyd-Laney, H.,Parkin, A.,Love, A.,George, M.W.,Pordea, A.,Morra, S.
Probing the Role of Accessory Domains in Oxygen Stability of [FeFe]-Hydrogenases.
J.Am.Chem.Soc., 148:29985-29996, 2026

PubMed Abstract: [FeFe]-hydrogenases are the fastest enzymes for hydrogen evolution, yet their irreversible inhibition by oxygen has thwarted their industrial use. A5H is an exception as its inhibition by oxygen is reversible. Protein scaffold rearrangement near the active site, allowing a ligand to coordinate the metal center in addition to the formation of a highly oxidized state of the metal center named H, is the current hypothesis for A5H oxygen stability. However, the ligand identity has been disputed and there is no evidence to suggest that protein scaffold rearrangement is the sole reason for oxygen stability. Here, we investigate A5H oxygen stability by providing a high-resolution (1.96 Å) X-ray structure that shows that the protective ligand is a conserved cysteine thiol group, which directly coordinates the metal center. The local rearrangement also encompasses structural water molecules and the side chain of E341, associated with proton transfer. In addition, we illustrate that C236 and H245, located close to accessory iron sulfur clusters in the Fd domain, influence oxygen stability. We show that mutating these residues significantly decreases oxygen stability but not the ability to form H. Variant C236A displays a slower inactivation rate, which we suggest is due to tuning the redox properties of one of the accessory iron sulfur clusters. We also show that the soluble ligand-binding β-grasp domain (SLBB) may not be required for oxygen stability by comparing A5H to a novel homolog lacking this domain. Collectively, these findings expand our understanding of oxygen stability in [FeFe]-hydrogenases.

PubMed: 42430233
DOI: 10.1021/jacs.6c05865

Experimental method

X-RAY DIFFRACTION (1.96 Å)