The H 2 Sensor of Ralstonia eutropha Is a Member of the Subclass of Regulatory [NiFe] Hydrogenases

Abstract

ABSTRACT Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus ) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H 2 , which is available in low amounts in aerobic environments. H 2 sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H 2 sensor of R. eutropha is a cytoplasmic protein. Although capable of H 2 oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H 2 sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231–235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H 2 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 subsequent H 2 oxidation and electron transfer processes are necessary for signaling. 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 denoted as the “regulatory hydrogenases.”