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Updated: Apr 4, 2026
![Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase](/_next/image?url=https%3A%2F%2Fcloudfront.jove.com%2FCDNSource%2Fteasers%2F55858.jpg&w=3840&q=50)
Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase
Published on: December 4, 2017
Formation of a Proton-Conducting Hydrogen-Bond Network during the L/M Transition of NsXeR Uncovered by Light-Induced
Yuma Ito1, Kirill Kovalev2, Tatsuro Nishikino1,3
1Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Abstract:
Light-driven inward proton pumping by xenorhodopsins (XeRs) challenges the view that proton-pumping microbial rhodopsins mainly generate outward proton gradients. A xenorhodopsin found from Nanosalina (NsXeR) exhibits particularly high activity, yet the determinants of efficient inward pumping remain unclear. We used temperature-dependent light-induced FTIR difference spectroscopy to probe L and M formation beyond the K intermediate at 77 K. In L, the Schiff-base N-D stretching modes shift markedly to lower frequency, indicating a strongly hydrogen-bonded Schiff base, while an intense water O-D band emerges, consistent with assembly of a multiwater network. The carboxylic C═O stretch of Asp220 shows intermediate-specific changes that distinguish L from M and implicate Asp220 in handling protons on the cytoplasmic side. Together, the spectra support a model in which a water-mediated hydrogen-bond network organized during L-to-M formation promotes rapid proton transfer in the cytoplasmic side, consistent with a Grotthuss-type mechanism.
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