Mass spectroscopic analysis of N 2 formation by flash-induced oxidation of hydrazine and hydroxylamine in normal and tris-treated tobacco chloroplasts

Abstract

The flash-induced dinitrogen evolution was analyzed in normal and Tris-inhibited tobacco chloroplasts ( Nicotiana tabacum var. John William's Broadleaf) which were dark-incubated (at least 20 min) in the presence of NH 2NH 2 and/or NH 2OH. (a) The N 2 yield due to a single turnover flash as a function of NH 2NH 2 concentration is practically the same in both normal and Tris-washed chloroplasts, even in a range where the oxygen evolution remains almost unaffected by NH 2NH 2. Illumination with ten flashes leads to higher integral N 2 evolution in Tris-washed samples at limiting NH 2NH 2 concentrations. (b) Under aerobic conditions oxidation of NH 2NH 2 and NH 2OH is coupled with significant O 2 uptake. Both N 2 formation and O 2 uptake remain unaffected by DCMU in a single turnover flash but become drastically inhibited under multiple flash excitation. (c) The N 2 yield per flash due to PS-II-induced NH 2NH 2 oxidation markedly depends on the O 2 content of the suspension. This effect is also observed in the presence of superoxide dismutase (SOD). (d) If chloroplasts are incubated with an equimolar ratio of 14NH 2 14NH 2 and 15NH 2 15NH 2, hardly any 14N 15N formation is detected, indicating that the nitrogen-nitrogen bond remains intact during the oxidation process. A totally different isotope distribution is obtained in the case of an equimolar mixture of 14NH 2OH and 15NH 2OH which reflects a statistical bimolecular reaction between NHOH radicals. In a mixture of 14NH 2OH and 15NH 2 15NH 2 the ratio of mass 29 to mass 30 increases with increasing 14NH 2OH content. Based on the present results, flash-induced N 2 formation is inferred to occur via univalent oxidation of NH 2OH and NH 2NH 2 either by P-680 + or by the functionally connected tyrosine(s) of polypeptide D-1 (Y ox Z) and/or D-2 (Y ox D). NH 2OH and NH 2NH 2 do not interact with the S 1 state of the catalytic site of water oxidation in a way that leads to flash-induced N 2 production.