Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

Abstract

(Ammonia plus 2-oxoglutarate)-dependent O2 evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9-15 μmol mg(-1) Chl h(-1). Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O2 evolution at rates of 6-11 μmol mg(-1) Chl h(-1) in the presence of MgCl2, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH4Cl, O2 evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH4Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O2 evolution was attributed to the synthesis of glutamine from NH4Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H2O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.