Biochemical Analysis of the Interactions between the Proteins Involved in the [FeFe]-Hydrogenase Maturation Process

The potential use of microorganisms for biological production of hydrogen as a future energy resource
makes hydrogen metabolism an emerging field of research. Hydrogenase (H2ase) is the name given
to the family of enzymes that catalyse the reversible oxidation of hydrogen into its elementary particle
constituents, two protons (H+) and two electrons. The reversible bioreduction of protons to molecular
H2 is catalyzed by [FeFe]-hydrogenases through the H-cluster active site, consisting of a [4Fe-4S]
cubane bridged to a 2Fe modified unit. The assembly of a catalytically competent H-cluster requires
three ancillary, highly conserved proteins: HydE, HydG and HydF. The latter functions as a scaffold
upon which HydG and HydE modify the bimetallic subcluster, which is then transferred by HydF to
HydA.
Through biochemical analysis (Heterologous Expression of Hyd Maturation and Structural Proteins
from Clostridium acetobutylicum, Co-purification of HydF with Potential Interaction Partners, Analysis
of the Stoichiometry of HydF-HydE and HydF-HydG-Interactions, and Purification of HydE and HydGProteins
to Homogeneity for Biacore Analysis), we provide evidences that interactions mediated by
the HydF scaffold function are essential and indipendent events, since HydF is able to interact with
both HydE and HydG in a binary way. We also suggest that, although HydF GTPase activity is likely modulated by the two other maturases, it seems not involved in the HydG and HydE binding
behaviour. The different oligomeric states in which HydF is found in vivo do not exhibit a differential
ability to associate with each of the two maturases, restricting useful data to identify the scaffold active
state of HydF. We finally tested the HydA-HydF interaction and suggest that HydG and HydE strongly
modify the HydF binding properties, since HydF becomes able to bind HydA only when expressed in
the presence of HydG and HydE.