[Fe-Fe] hydrogenases, with hydrogen evolution activities outperforming [Ni-Fe] hydrogenases by 3-4 orders of magnitude, are still the most promising enzyme class for hydrogen production purposes. For Chlamydomonas reinhardtii [Fe-Fe] hydrogenase HydA1 the question of catalytic activity and electron transport is of main importance. Here we report the characterization of two mutant forms of C. reinhardtii HydA1. An aspartic acid in place of arginine(171) leads to a six-fold increase of the catalytic activity in comparison to the wild type protein during methyl viologen-dependent hydrogen production. Tryptophan in position 171 does not result in any change in methyl viologen-induced activity. At the same time these mutations lead to a strong decrease in ferredoxin-dependent hydrogen production while the catalytic center of mutant forms stays intact. The localization of this amino acid (arginine(171)) in the environment of CrHydA1 H-cluster indicates that the limitation of the catalytic activity of this hydrogenase is due to the electron transfer step to the catalytic center where the reduction of protons takes place. Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.