Although of potential biotechnological interest, photobiological H-2 production from microalgae and cyanobacteria is strongly limited due to the oxygen sensitivity of hydrogenases, the H-2-evolving enzymes. We study here the [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. FCC 6803 to identify structural determinants of its sensitivity to O-2. Based on previous work on the hydrogenase from Desulfovibrio fructosovorans and on a structural model of the Synechocystis hydrogenase, we have created various mutants of the Synechocystis enzyme. Amino acids residues homologous to those defining the end of the intramolecular gas channel in the D. fructosovorans enzyme were specifically targeted, these residues being previously described as critical for enzyme activity and tolerance to O-2. We show here that mutation I64M of the Synechocystis enzyme alters gas diffusion kinetics and improves O-2 tolerance. These results constitute the first report demonstrating that an O-2 tolerance-related character could be transposed from a proteobacterial hydrogenase to a cyanobacterial one, and may constitute the first published improvement of O-2 tolerance of a cyanobacterial enzyme by single site-directed mutagenesis. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.