Critical Residues of Chlamydomonas reinhardtii Ferredoxin for Interaction with Nitrite Reductase and Glutamate Synthase Revealed by Site‐Directed Mutagenesis

• Published in: European Journal of Biochemistry Vol. 250; no. 2; pp. 364 - 368
• Main Authors: García‐Sánchez, M. Isabel, Gotor, Cecilia, Jacquot, Jean‐Pierre, Stein, Mariana, Suzuki, Akira, Vega, José M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.12.1997; Wiley
• Subjects: Animals; binding domain; Binding Sites; Biochemistry, Molecular Biology; Chlamydomonas - enzymology; Chlamydomonas reinhardtii; ferredoxin; Ferredoxins - chemistry; Freshwater; glutamate synthase; Glutamate Synthase - chemistry; Life Sciences; Mutagenesis, Site-Directed; nitrite reductase; Nitrite Reductases - chemistry; site‐directed mutagenesis
• ISSN: 0014-2956; 1432-1033; 1432-1327
• DOI: 10.1111/j.1432-1033.1997.0364a.x

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 α1 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 photo synthetic chain.