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

<jats:p> Hydrogenases catalyze the reversible formation of H <jats:sub>2</jats:sub> , and they are key enzymes in the biological cycling of H <jats:sub>2</jats:sub> . H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H <jats:sub>2</jats:sub> 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 <jats:sub>2</jats:sub> 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 <jats:sub>2</jats:sub> . Using our new approach, we determined that the fractionation factor for H <jats:sub>2</jats:sub> production by the [NiFe]‐hydrogenase from <jats:italic>Desulfovibrio fructosovorans</jats:italic> is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H <jats:sub>2</jats:sub> evolution. Potential applications of this newly developed method are discussed. Copyright © 2011 John Wiley & Sons, Ltd. </jats:p>