Biological H-2 production is one of the alternative technologies for producing H-2 in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H-2 production reaction from protons and electrons as it is highly efficient. The high O-2 sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O-2 tolerance via protein engineering. In this study, we investigated the changes in the O-2 tolerance and aerobic H-2 production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O-2 tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O-2 tolerance with the rest of the Met - Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O-2 tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.