Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex

Abstract

Nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) of Chlamydomonas reinhardii mutant 305. lacking NAD(P)H-cytochrome c reductase activity, became rapidly and reversibly inactivated in vitro upon incubation with dithionite and reactivated by oxidation with ferricyanide. Nitrate protected against the inactivation. Unlike the native nitrate reductase complex of its parental wild strain, the enzyme of 305 was not inactivated by reduced pyridine nucleotides. In contrast to that of wild-type cells, the in vivo reversible inactivation of mutant 305 nitrate reductase was observed only after complete elimination of nitrate from the media and subsequent transfer to ammonium medium or darkness conditions. The inactive enzyme was reactivated by addition of nitrate to the media without previous removal of ammonium, which indicates that, unlike in the wild-type cells, ammonium does not prevent nitrate uptake by 305 cells. This different regulation pattern is due to the structural modification of 305 nitrate reductase. We conclude that in vitro an active diaphorase moiety is required for the inactivation by reduced pyridine nucleotides of the nitrate reductase of C. reinhardii, and that in vivo the absence of nitrate rather than the presence of ammonium is the triggering event for nitrate reductase inactivation, which can be also achieved by reductants other than NAD(P)H.