Hydrogenases are proteins which catalyze the reversible two-electron oxidation of the most simple of chemical compounds, molecular hydrogen, following the reaction: H2 ⇔ 2H++2e−. These enzymes are the central feature of hydrogen metabolism which is essential to many microorganisms of great biotechnological interest, such as methanogenic, acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria. For the last twenty years hydrogenases have enjoyed renewed interest, mostly in view of their capacity to produce molecular hydrogen, a clean source of energy.
The determination of the first three-dimensional atomic structure of a hydrogenase by single crystal X-ray diffraction, together with infrared (IR) and electron paramagnetic resonance (EPR) spectroscopy have revealed that the active site consists of a bimetallic center containing for [NiFe] hydrogenases one nickel and one iron atom and three non-protein diatomic ligands (one CO and two CN−) to the iron. This evidence with stoichiometric redox-titrations has led to new insights into the catalytic mechanism. Moreover, genetic studies have cast light on the biosynthesis of the protein, including the incorporation of metal atoms found at the active site.
A brief overall view of the structural and functional properties of hydrogenase is presented. The crystal structure is described with emphasis on the determination of the atomic content and arrangement of the active site from combined X-ray and spectroscopic data. Some aspects of the catalytic mechanism are discussed in the light of these new results.