Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H 2 production by hydrogenases

Hydrogenases catalyze the reversible formation of H 2 , and they are key enzymes in the biological cycling of H 2 . H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H 2 production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme‐catalyzed H 2 evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom‐designed high‐throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H 2 . Using our new approach, we determined that the fractionation factor for H 2 production by the [NiFe]‐hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H 2 evolution. Potential applications of this newly developed method are discussed. Copyright © 2011 John Wiley & Sons, Ltd.