Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H2 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.
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Hui
H
Yang
Hasand
H
Gandhi
Liang
L
Shi
Helen W
HW
Kreuzer
Nathaniel E
NE
Ostrom
Eric L
EL
Hegg
D003901:Desulfovibrio / Q000201:enzymology; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006859:Hydrogen / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism*; D057230:Limit of Detection; D011522:Protons
D011522:Protons; D006859:Hydrogen; D003903:Deuterium; C043296:nickel-iron hydrogenase; D006864:Hydrogenase