The H-2 sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ra lstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-contai ning enzymes has to be efficiently regulated in response to H-2, which is a vailable in low amounts in aerobic environments. H-2 sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase g ene expression in concert with a two-component regulatory system. In this s tudy we show that the H-2 sensor of R. eutropha is a cytoplasmic protein. A lthough capable of H-2 oxidation with redox dyes as electron accepters, the protein did not support lithoautotrophic growth in the absence of the ener gy-generating hydrogenases. A specifically designed overexpression system f or R. eutropha provided the basis for identifying the H-2 sensor as a nicke l-containing regulatory protein. The data support previous results which sh owed that the sensor has an active site similar to that of prototypic [NiFe ] hydrogenases (A. J. Pierik, M. Schmelz, O. Lent, B. Friedrich, and S. P. J. Albracht, FEES Lett. 438:231-235, 1998). It is demonstrated that in addi tion to the enzymatic activity the regulatory function of the R, sensor is nickel dependent. The results suggest that H-2 sensing requires an active:[ NiFe] hydrogenase, leaving the question open whether only H-2 binding or su bsequent H-2 oxidation and electron transfer processes are necessary for si gnaling. The regulatory role of the H-2-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H-2 sensors represents a novel subclass of [NiFe] hydrogenases de noted as the "regulatory hydrogenases."