The [FeFe]-hydrogenase of Rhodospirillum rubrum (Rr-HydA; Rru_A0310) was expressed in an Escherichia coil strain co-expressing the hydrogenase maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum (Ca-HydEFG). Rr-HydA, which purified as a 50 kDa protein, showed an in vitro H-2-evolving activity in the presence of dithionite-reduced methyl viologen. The ability of Rr-HydA to carry out H-2-evolving activity when heterologously expressed in a Rhodobacter sphaeroides nifDK hupSL mutant containing the genes coding for Ca-HydEFG was examined. Photoheterotrophic H-2 evolution was only observed in the presence of Rr-HydB [Rru_A0309 (22 kDa)], which is encoded by a gene located immediately upstream from the gene encoding Rr-HydA. Rr-HydB contains Fe-S cluster(s). Consistently, in a reaction mixture containing spinach ferredoxin oxidoreductase and NADPH, H-2 was evolved when both Rr-HydA and Rr-HydB were present together, and the rate of H-2 evolution by Rr-HydA was proportional to the level of Rr-HydB until an equimolar ratio of the two proteins was reached. Thus, it is proposed that Rr-HydB acts as Rr-HydA-specific ferredoxin that donates electrons to Rr-HydA through a direct molecular interaction. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.