[FeFe]-Hydrogenases, encoded by hydA are thought to require the products of three accessory genes, hydE, F and G, for the biosynthesis of a functional H-cluster and maturation into a functional protein capable of reducing protons to hydrogen. Genome sequencing has shown that some organisms, including several strains of purple non-sulfur photosynthetic bacteria, possess orphan hydAs. It is unknown if these orphan HydAs can be matured into functional proteins. A construct where transcription of the apparent orphan hydA of Rhodospirillum rubrum was driven by the nifH2 promoter failed to restore hydrogen production to a Nif(-) strain of R. rubrum under nitrogen-limited photo-heterotrophic conditions. However, RrhydA could be overexpressed and matured in Escherichia coli BL21(DE3) containing the hydE, F and G maturation genes from Desulfovibrio vulgaris Hildenborough or Clostridium acetobutylicum ATCC 824. Co-expression of R. rubrum hydA with the maturation genes of C. acetobutylicum gave the highest hydrogenase activity, 107 nmol/min/mg, whereas it was 6.6 nmol/min/mg with the maturation genes from D. vulgaris. Interestingly, R. rubrum HydA was twice as active as the HydA of C. acetobutylicum when both were matured with the maturases from the latter organism. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.