THE AMINO ACID SEQUENCE OF SPINACH FERREDOXIN

Ferredoxins are a group of non heme iron-containing proteins present in certain anaerobic nonphotosynthetic bacteria' and in all photosynthetic organisms.2 The amino acid sequences of two nonphotosynthetic bacterial ferredoxins obtained from Clostridium pasteurianum and Clostridium butyricum have an unusual pattern. They contain essentially repeating sequences of a peptide containing 22 amino acid residues, four of which are cysteines,3-7 and they are free from arginine, histidine, leucine, methionine, and tryptophan. The unusual structure of bacterial ferredoxins stimulated interest in the evolutionary development of this type of protein.5-7 Ferredoxins have been divided into two types, a bacterial and a plant type that share certain properties but differ in others.2 The amino acid sequence of plant ferredoxin is of considerable interest from the standpoint of evolution and the relation of protein structure to function. In preliminary investigations, Tsugita et al.8 and Fry and San Pietro9 reported that spinach ferredoxin is high in acidic and low in basic amino acids, and that it contains single residues of arginine, histidine, methionine, and tryptophan. Tsugita et al. also found that the aminoand carboxyl-terminal amino acids were alanine8 and that the carboxyl-terminal tripeptide sequence was Leu-Thr-Ala.'0 This article reports the complete amino acid sequence of spinach ferredoxin. Materials and Methods.-Ferredoxin was prepared from spinach leaves, obtained commercially, according to the method of Tagawa and Arnon."1 These preparations had Emo/E276 = 0.43 0.46 and were used without further purification. The molecular weight of spinach ferredoxin was determined by the gel filtration method'2 on Sephadex G-75, by amino acid analysis,'3 and by analyses of the terminal residues using modified Edman degradation'4 and carboxypeptidase A procedures."5 Prior to the sequential study the protein was treated with trichloroacetic acid.'6 S-3-aminoethylcysteinyl-ferredoxin (AEFd), S-catboxymethylcysteinyl-ferredoxin (CMOFd), and oxidized ferredoxin (OFd) were prepared as described 17-19 with slight modifications. The amino-terminal sequence was determined by the stepwise Edman degradation procedures with a slight modification in the case of intact ferredoxin, OFd, and CMFd. The carboxyl-terminal sequence was determined by the carboxypeptidase A procedure' on intact and carboxymethylated ferredoxins. The peptide fragments were obtained by digestion of various derivatives of ferredoxin with trypsin, chymotrypsin, and thermolysin. (Thermolysin is available from Chugai Boyeki Co., Ltd., P.O. Box Higashi no. 106, Osaka, Japan.) About 15 imoles of AEFd were hydrolyzed at pH 8.2 and 25° with 3 mg of trypsin for 1.5 hours and for a further 3 hours with an additional 2 mg of trypsin. The pH was adjusted to 4.5 with acetic acid, and the resultant precipitate was removed by centrifuging. The supernatant solution was lyophilized. The separation of the peptides was carried out on an analytical-grade cation exchange column, Bio-Rad