Mass spectrometric analysis of the light-induced hydrogen gas exchange in the cyanobacteria Oscillatoria chalybea, Synechocystis PCC 6803, Synechococcus PCC 6301 (Synechococcus leopoliensis; Anacystis nidulans) and Synechococcus elongatus has been carried out by direct detection of molecular hydrogen at m/e = 2 in the HID collector of a 'delta' mass spectrometer. The time curves of the signals reveal an initial outburst of hydrogen at the onset of light, with a subsequent superimposition of a hydrogen uptake in the case of Oscillatoria chalybea. With in vivo cultures of Synechocystis PCC 6803, which have not been shown to photoevolve molecular hydrogen so far, we are able to measure very small but clearly detectable evolution and uptake signals. The principal qualitative features of the transition from hydrogen evolution to uptake during the illumination period in the cases of Oscillatoria chalybea and Synechocystis PCC 6803 are about identical, Upon addition of increasing concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), an increased stable hydrogen photoevolution shows up with the hydrogen uptake being completely suppressed. In the case of Oscillatoria chalybea this effect is obtained with 5 mu M CCCP, whereas with Synechocystis PCC 6803 generally higher CCC concentrations are required to stabilize the hydrogen evolution over time. However, in the presence of CCCP the hydrogen evolution from Synechocystis PCC 6803 (which is nearly negligible in the controls without additions) reaches values very similar to those observed with Oscillatoria chalybea. The stimulatory effect of CCCP is clearly distinct from an uncoupling of electron transport and photophosphorylation. as the addition of ammonium chloride inhibits hydrogen evolution but exerts the expected effect of stimulation of photosynthetic oxygen evolution. However, modification of the pH value of the reaction buffer results in a clear dependency of the hydrogen gas exchange on the external proton concentration. Basic pH values at about >9 diminish the gas exchange signal as a whole. Acidic pH at about 4-5 substantially increases the evolution and decreases the uptake part of the gas exchange signals, Thus, ar a pH of 4.4, the hydrogen gas exchange signal largely corresponds to the one observed with a assay at pH 7.5 but in the presence of CCCP, When the cyanobacterial reaction assays are flushed with pure nitrogen or with N-2/CO2 (1%), the hydrogen evolution rates are by no means decreased; in some cases the hydrogen photoevolution appeals rather to be stimulated by the presence of CO2 in the flushing gas. Concomitant recording of the light-induced carbon dioxide reveals a strong initial CO2 uptake which is substantially diminished within the first minute of illumination. Thus, mass spectrometric analysis of the light-induced carbon dioxide gas exchange favours the CO2 concentrating mechanism discussed for cyanobacteria rather than the light activation of a dark inactive Calvin cycle. (C) 1998 Elsevier Science S.A. All rights reserved.
