Isolation and characterisation of a new [FeFe]-hydrogenase from Clostridium perfringens.

This paper reports the first characterisation of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant, that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulphur centres involved in electron transfer and the catalytic centre H-cluster. After recombinant expression in E. coli, the purified protein catalyses H2 evolution at high rate of 1645±16 s−1. The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50°C. EPR spectroscopy showed that the H-cluster of the oxidised enzyme displays a spectrum coherent with the Hox state, while the CO inhibited enzyme has a spectrum coherent with the Hox-CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox; upon reduction with H2, vibrational modes assigned to the Hred state were more abundant, while binding of exogenous CO resulted in a spectrum assigned to the Hox-CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster.