CRITICAL RESIDUES OF CHLAMYDOMONAS-REINHARDTII FERREDOXIN FOR INTERACTION WITH NITRITE REDUCTASE AND GLUTAMATE SYNTHASE REVEALED BY SITE-DIRECTED MUTAGENESIS

Incubation of wild-type ferredoxin (Fd) with Chlamydomonas reinhardtii crude extract in the presence of a carboxyl activator resulted in the formation of a unique 1:1 covalent complex with nitrite reductase. Ho wever, glutamate synthase was able to form two covalent complexes of F d:GOGAT with 1:1 and 2:1 stoichiometries. These complexes were functio nal only when reduced methyl viologen was used as electron donor. Kine tic studies of complex formation suggested the presence of two Fd-bind ing domains with similar affinity for Fd in the glutamate synthase mol ecule. Using site-directed mutagenesis with recombinant Fd, we have sh own that Fd-Glu91 is directly involved in Fd interaction with glutamat e synthase and nitrite reductase. Moreover, a negative core of residue s in the al helix of Fd was also critical in binding the enzymes. Thes e data highlight the analogy in the Fd-binding sites of nitrite reduct ase and glutamate synthase, which may compete for the electrons coming from the photosynthetic chain.