The amino acid sequence of Leucaena glauca ferredoxin

Abstract

Ferredoxin, a protein of unusually low oxidation-reduction potential, and also the first protein found to contain both iron and labile sulfide, plays a very fundamental role in the photosynthetic process, that of acceptor of electrons from light-activated chlorophyll. Ferredoxin was isolated from the leaves of Leucaena glauca, a species of small leguminous tree. The carboxy-methylcysteine derivative of the combined ferredoxin from many plants was used for the amino acid sequence studies. The products of tryptic and chymotryptic hydrolyses were purified by ion exchange chromatography, gel filtration, and paper chromatography. Thermolysin and Bacillus subtilis neutral protease were used to further degrade large peptides. Most of the sequence was determined by the subtractive method of Edman degradation (J. Biol. Chem., 237, 2547, 1962). The amino acid sequence, deduced primarily from the structures of the tryptic and chymotryptic peptides, is as follows: Ala-Phe-Lys-Val-Lys-Leua/Valb-Leu-Thr-Pro-Asp-Gly-Proa/Alab-Lys-Glu-Phe-Glu-Cys-Pro-Asp-Asp-Val-Tyr-Ile-Leu-Asp-Gln- Ala-Glu-Glu-Leu-Gly-Ile-Glux/Aspx-Leu-Pro-Tyr-Ser-Cys-Arg-Ala- Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Leu-Val-Glu-GIy-Asp-Leu- Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Glu-Gln-Ile-Glu-Glu- Gly-Trp-Val-Leu-Thr-Cys-Ala-Ala-Tyr-Pro-Arg-Ser-Asp-Val-Val- Ile-Glu-Thr-His-Lys-Glu-Glu-Glu-Leu-Thr-GlYx/Alax-COOH. The sequence of this ferredoxin resembles those of spinach and alfalfa ferredoxins, showing a slightly greater similarity to alfalfa, as would be expected from taxonomic considerations. Over 70% of the sequence, including the five cysteine residues, is invariant in these three species. Many of the changes between species are conservative, but radical changes involving charged residues and proline residues occur also. Heterogeneity was found in positions 6, 12, 33, and 96 within the L. glauca ferredoxin sequence, each of which was occupied by two different amino acid residues, as shown. However, the tryptic peptide containing residues 6 and 12 occurred in only two of the four possible forms, one of which contained leucine and proline in positions 6 and 12, respectively, whereas the other contained valine and alanine. Thus the presence of different genes is indicated, rather than ambiguity in translation of the genetic code. A study of the distribution of the different forms of the protein among the ~. glauca population was undertaken. In a normal distribution of allelic genes, 50% of the population would be expected to be homozygous. Ferredoxin was isolated from ten different plants and three of the four points of heterogeneity were investigated (residues 6, 12, and 96). Heterogeneity was found in each of these positions in each of the ten individual trees. Thus the probability that the differing allelic nuclear genes are involved is less than 0.001. The two most probable explanations for the observed sequence heterogeneity are (1) that differing non-allelic genes for ferredoxin are present, that is, that gene duplication has occurred in the evolution of this species, followed by point mutations in the individual genes, or (2) that the ferredoxin genes are located not in the nucleus of the call but rather in the chloroplasts, and their distribution therefore follows a pattern quite different from that of allelic nuclear genes.