title: Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens.
abstract: 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 H2 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 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 oxidized enzyme displays a spectrum coherent with the Hox state, whereas 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, whereas 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.
authors: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors: Simone
authors: S
authors: Morra
authors: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors: Beatrice
authors: B
authors: Mongili
authors: Department of Chemistry, University of Torino, Torino, Italy.
authors: Sara
authors: S
authors: Maurelli
authors: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors: Gianfranco
authors: G
authors: Gilardi
authors: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors: Francesca
authors: F
authors: Valetti
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D017421:Sequence Analysis
chemical_list: D006864:Hydrogenase
keywords: Clostridium perfringens; H-cluster; [FeFe]-hydrogenases; bio-hydrogen; iron-sulfur centers; recombinant expression