title: Critical residues of Chlamydomonas reinhardtii ferredoxin for interaction with nitrite reductase and glutamate synthase revealed by site-directed mutagenesis.
abstract: 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.
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC y Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja, Spain.
authors.forename: M I
authors.initials: MI
authors.lastname: García-Sánchez
authors.forename: C
authors.initials: C
authors.lastname: Gotor
authors.forename: J P
authors.initials: JP
authors.lastname: Jacquot
authors.forename: M
authors.initials: M
authors.lastname: Stein
authors.forename: A
authors.initials: A
authors.lastname: Suzuki
authors.forename: J M
authors.initials: JM
authors.lastname: Vega
mesh_terms: D000818:Animals; D001665:Binding Sites; D002696:Chlamydomonas / Q000201:enzymology*; D005288:Ferredoxins / Q000737:chemistry*; D005970:Glutamate Synthase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry*
chemical_list: D005288:Ferredoxins; D005970:Glutamate Synthase; D009572:Nitrite Reductases