A histidine-mediated, pendulum-like proton transport mechanism is required for the high catalytic activity of -hydrogenases
- 1Ruhr University Bochum
Abstract
[FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.
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BibTeX
@misc{duan2026histidinemediated,
title = {{A histidine-mediated, pendulum-like proton transport mechanism is required for the high catalytic activity of -hydrogenases}},
author = {J. Duan and Z. Yang and O. Lampret and F. Arrigoni and L. V. Impel and F. Zheng and J. Kleinhaus and U. P. Apfel and C. Greco and L. D. D. Gioia and E. Hofmann and A. Hemschemeier and T. Happe},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.22.753672},
url = {https://www.biorxiv.org/content/10.64898/2026.09.22.753672v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - A histidine-mediated, pendulum-like proton transport mechanism is required for the high catalytic activity of -hydrogenases
AU - J. Duan
AU - Z. Yang
AU - O. Lampret
AU - F. Arrigoni
AU - L. V. Impel
AU - F. Zheng
AU - J. Kleinhaus
AU - U. P. Apfel
AU - C. Greco
AU - L. D. D. Gioia
AU - E. Hofmann
AU - A. Hemschemeier
AU - T. Happe
PY - 2026
DA - 2026/09/22
PB - bioRxiv
DO - 10.64898/2026.09.22.753672
UR - https://www.biorxiv.org/content/10.64898/2026.09.22.753672v1
AB - [FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.
ER -