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

This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H-2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H-2 evolution at high rate of 1645 +/- 16s(-1). The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50 degrees C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the H-ox state, whereas the CO-inhibited enzyme has a spectrum coherent with the H-ox-CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the H-ox; upon reduction with H-2, vibrational modes assigned to the H-red state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the H-ox-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.