Involvement of hyp gene products in maturation of the H-2-sensing [NiFe] hydrogenase of Ralstonia eutropha

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and Hy pX for assembly of the H-2-activating [NiFe] site. In this study we examine d the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H-2-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [Ni Fe] hydrogenases. Here we demonstrate that the RE regulatory activity was b arely affected by mutations in hypA, hypB, hypC, and hypX and was not subst antially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of Ni-63 into the RH from overproducing cells revealed that the assembly of the [NiFe] active si te is dependent on all Hyp functions, with the exception of HypX. We conclu de that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) i s involved in an efficient incorporation of the [NiFe] center into the RH.