A GAS-CHROMATOGRAPHIC MASS-SPECTROMETRIC TECHNIQUE FOR STUDYING SIMULTANEOUS HYDROGEN DEUTERON EXCHANGE AND PARA ORTHOHYDROGEN CONVERSION IN HYDROGENASES OF DESULFOVIBRIO-VULGARIS HILDENBOROUGH

An original gas chromatographic-mass spectrometric technique is described for studying simultaneous dihydrogen-deuteron exchange and para-ortho H2 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 H2 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 H2), m e 3 (HD), and m e 4 (D2) 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 D2O. These findings are related to the constitutional and catalytic properties of the hydrogenases belonging to the different classes. © 1990.