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. However, glutamate synthase was able to form two covalent complexes of Fd: GOGAT with 1:1 and 2:1 stoichiometries. These complexes were functional only when reduced methyl viologen was used as electron donor. Kinetic studies of complex formation suggested the presence of two Fd-binding domains with similar affinity for Fd in the glutamate synthase molecule. Using site-directed mutagenesis with recombinant Fd, we have shown that Fd-Glu91 is directly involved in Fd interaction with glutamate synthase and nitrite reductase. Moreover, a negative core of residues in the alpha1 helix of Fd was also critical in binding the enzymes. These data highlight the analogy in the Fd-binding sites of nitrite reductase and glutamate synthase, which may compete for the electrons coming from the photosynthetic chain.