9RJO

[FeFe]-hydrogenase CpI from Clostridium pasteurianum, variant N160L-Q195L

Summary for 9RJO

• Entry DOI: 10.2210/pdb9rjo/pdb
• Descriptor: Iron hydrogenase 1, dicarbonyl[bis(cyanide-kappaC)]-mu-(iminodimethanethiolatato-1kappaS:2kappaS)-mu-(oxomethylidene)diiron(2+), IRON/SULFUR CLUSTER, ... (8 entities in total)
• Functional Keywords: [fefe]-hydrogenase i from clostridium pasteurianum, variant n160l-q195l, oxidoreductase
• Biological source: Clostridium pasteurianum
• Total number of polymer chains: 2
• Total formula weight: 134417.97

Authors

Duan, J.,Liu, L.,Hofmann, E.,Happe, T. (deposition date: 2025-06-12, release date: 2026-03-04, Last modification date: 2026-04-01)

Primary citation

Liu, L.,Klamke, M.A.,Arrigoni, F.,Lampret, O.,Kleinhaus, J.,Apfel, U.P.,Hofmann, E.,Greco, C.,Happe, T.,Stripp, S.T.,Duan, J.
A Zundel ion in the catalytic proton transfer pathway of [FeFe]-hydrogenase.
Phys Chem Chem Phys, 28:7101-7110, 2026

PubMed Abstract: [FeFe]-hydrogenases are metalloenzymes that catalyze the interconversion of protons, electrons, and molecular hydrogen (H). Their active site cofactor consists of a [4Fe-4S] cluster ([4Fe]) and a diiron site ([2Fe]), forming the so-called H-cluster. In this work, the putative regulatory proton transfer pathway (PTP) toward the [4Fe] cluster of [FeFe]-hydrogenase I from is characterized by X-ray crystallography, infrared spectroscopy, and quantum mechanical (QM) calculations. The trajectory consists of asparagine N160, glutamine Q195, and several protein-bound water molecules that might function as a PTP toward cysteine C499 at the [4Fe] cluster. We have hypothesized that protonation of C499 determines the H-cluster intermediate HH (M. Senger , , 2018, 20, 3128-3140). The crystal structures of protein variants N160L and Q195L now confirm that the putative regulatory PTP is disrupted. However, infrared spectroscopy reveals that all variants accumulate the HH state in a manner comparable to wild-type I. In contrast, the I variant E279D - previously shown to target the catalytic PTP toward [2Fe] - is found to enrich the HH state independently of reducing agents. This indicates that the determinants of HH are located in the catalytic PTP, which emphasizes the importance of HH during catalysis and provides evidence against any involvement of the putative regulatory PTP in hydrogen turnover. Supported by QM calculations, a model is proposed in which a conserved water cluster adjacent to E279 is protonated to form a Zundel ion (HO). Our results paint a new picture of the H-cluster in the HH state and yield important insight into the catalytic mechanism of [FeFe]-hydrogenases.

PubMed: 41774038
DOI: 10.1039/d5cp04267d

Experimental method

X-RAY DIFFRACTION (1.67 Å)