Authors
Mahapatra, G. P., Strabel, N., Lorent, C., Kumar, A., Bohn, S., Klamke, M. A., Boehm, M., Teutloff, C., Zebger, I., Appel, J., Schuller, J., Gutekunst, K.
Abstract
Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from dark carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 28 Jun 2026.
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