A gas chromatographic-mass spectrometric technique for studying simultaneous hydrogen-deuteron exchange and para-orthohydrogen conversion in hydrogenases of Desulfovibrio vulgaris Hildenborough

Abstract

An original gas chromatographic-mass spectrometric technique is described for studying simultaneous dihydrogen-deuteron exchange and para-ortho H 2 conversion catalyzed by different Desulfovibrio hydrogenases. Para and orthohydrogens are separated on an alumina column at the temperature of liquid nitrogen, but if both HD and ortho H 2 are present, their retention times are too close to each other for total separation and only one peak is observed with a thermal conductivity detector. In order to resolve the peaks from one another, a fraction of the gas released from the gas chromatograph column is admitted to the ion source of a mass spectrometer, where the gases are separated according to their respective masses. Because of a peak-jumping system, the different components involved in the exchange and in the conversion reactions can be scanned so that the spectra corresponding to mass m e 2 (para and ortho H 2), m e 3 (HD), and m e 4 (D 2) can be obtained simultaneously. This technique has been employed to resolve a controversial problem concerning the occurrence or lack of any para-orthohydrogen conversion in heavy water. Actually both exchange and conversion were demonstrated to occur with a (NiFe) hydrogenase, whereas with a (NiFeSe) hydrogenase, which had an exchange activity equivalent to that of the former, practically no para-ortho conversion could be observed in D 2O. These findings are related to the constitutional and catalytic properties of the hydrogenases belonging to the different classes.