Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis.
Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.
Gadda
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G
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Aliverti
A
A
Ronchi
S
S
Zanetti
G
G
D000595:Amino Acid Sequence; D002872:Chromosome Deletion; D005287:Ferredoxin-NADP Reductase / Q000037:antagonists & inhibitors / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation
D010446:Peptide Fragments; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D010447:Peptide Hydrolases