Donation of electrons to plastoquinone by NAD(P)H dehydrogenase and by ferredoxin-quinone reductase in spinach chloroplasts

The reduction of plastoquinone by NADPH was detected as an increase in the dark level of Chl fluorescence in osmotically ruptured chloroplasts of spinach. This activity was observed only when the chloroplasts were ruptured in a medium containing a high concentration of MgCl2. The activity was suppressed by inhibitors of the respiratory NADH dehydrogenase (NDH) complex in mitochondria, capsaicin and amobarbital, suggesting that the activity was mediated by chloroplastic NDH complex. Antimycin A, an inhibitor of ferredoxin-quinone reductase (FQR), and the protonophore nigericin also inhibited the increase in Chi fluorescence by NADPH, By contrast, N-ethylmaleimide (NEM), an inhibitor of ferredoxin-NADP(+) reductase (FNR), did not suppress the fluorescence increase, showing that FNR is not involved in this reaction. When the osmotically ruptured chloroplasts were washed by centrifugation, a further addition of ferredoxin as well as NADPH was required for an increase in fluorescence. This ferredoxin-dependent activity also was suppressed by antimycin A, but only partly inhibited by capsaicin or amobarbital, suggesting that this is mediated mainly by FQR. These findings suggest that the NADPH-binding subunit of NDH complex is easily dissociated from the thylakoid membranes during the process of the washing the thylakoids by centrifugation.