MOLECULAR BIOLOGY OF HYDROGENASES Claudio Tosi, Elisabetta Franchi, Francesco Rodriguez, Alessandro Selvaggi, and Paola Pedroni Eniricerche S.p.A. Environmental Technologies Via F. Maritano, 26-20097 San Donato Milanese, Italy Key Words hydrogenase, [Fe-SI cluster, hydrogenase gene cluster, Pyrococcus furiosus, Acetomicro- bium flavidum, Rhodobacter sphaeroides RV l. SUMMARY Hydrogenases, found in a wide variety of organisms, catalyze either the consump- tion or the production of H, in response to different physiological conditions. In recent years, a large body of biochemical and genetic data on enzymes isolated from different sources has contributed to elucidating fundamental aspects about their catalytic properties and gene organization. In addition, the recently obtained crystal strutture of a [Ni-Fe] hy- drogenase sheds iight on structure-function relationships in these enzymes. With the ulti- mate goal of engineering photosynthetic strains to improve their light-dependent hydrogen evolution capacity, we have characterized at the molecular leve1 hydrogenases from differ- ent microbial sources. In particular, we have focused our attention on the H,-evolving hy- drogenases from Pyrococcus furiosus and Acetomicrobium flavidum and on the uptake hydrogenase system from Rhodobacter sphaeroides RV. This rnicroorganism represents the species selected for use in a photobioreactor, as planned in the Japanese hydrogen pro- duction project in which we are involved. 2. INTRODUCTION Hydrogenases are a heterogeneous group of redox enzymes thae reversibly catalyze the prodetction or consumptioa of hydrogen according to the equakion: H, o 2H' i- 2e-. Bio-drogen, edttsd by Zaborcky et iab. Plenum Presc, New Vork, 1998