THE ROLE OF AROMATIC AND ACIDIC AMINO-ACIDS IN THE ELECTRON-TRANSFER REACTION CATALYZED BY SPINACH FERREDOXIN-DEPENDENT GLUTAMATE SYNTHASE

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mo l of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or red uced methyl viologen serving as the source of elections. The inhibitio n of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatmen t had no effect on the secondary structure of the enzyme. did not affe ct the K(m)s for 3-oxoglutarate and glutamine. did not affect the midp oint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that th e ferrodoxin-binding site(s) of glutamate synthase contains at least o ne, and possibly two. tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PC C 7120, with a non-aromatic amino acid, or replacement of the glutamat e at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the c hange at position 65 was quite modest compared to that at position 94. suggesting that an aromatic amino acid is not absolutely essential at position 65. but that glutamate 94 is essential for optimal electron transfer. (C) 1998 Elsevier Science B.V.