O-tolerant [NiFe]-hydrogenases of Ralstonia eutropha H16: Physiology, molecular biology, purification, and biochemical analysis.

Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H⇌2H+2e even in the presence of O. Catalytic and probably also noncatalytic mechanisms protect their active sites from being inactivated by reactive oxygen species, which makes them attractive subjects of investigation from both fundamental and applied perspectives. Prominent representatives of the O-tolerant [NiFe]-hydrogenases have been isolated from the chemolithoautotrophic model organism Ralstonia eutropha H16, which can thrive in a simple mineral medium supplemented with the gases H, O, and CO. In this chapter, we describe methods for cultivation and genetic manipulation of R. eutropha, both of which are prerequisites for the reproducible manufacturing of high-quality hydrogenase preparations. The purification procedures for two different O-tolerant [NiFe]-hydrogenases from R. eutropha are described in detail, as well as the corresponding biochemical procedures used for the determination of the catalytic properties of these sophisticated enzymes.