The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis.

The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.

Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.

Ferredoxin from a red alga, Porphyra umbilicalis.

A plant-algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non-haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was -400 mV and it effectively mediated electron transport in the NADP-photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lys4, His2, Arg1, Asx10, Thr8, Ser7, Glx16-17, Pro3, Gly7, Ala8, Cys5, Val6, Met1, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentation-equilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.

Studies on the small subunit of fraction I protein from Pisum sativum L. and Vicia faba L.

We have measured the molecular weight of the small subunit of Fraction I protein from pea and broad bean by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Sephadex gel-filtration and amino acid composition data. The results suggest a molecular weight of 12 000-14 500, although measurements by gel-filtration in alkali suggest a molecular weight of approximately 22 000. N-terminal amino acid sequence data and C-terminal determinations show that the protein consists of a single type of polypeptide chain, although the anomalously high molecular weight obtained on gel-filtration in alkali does not preclude the existence of the polypeptides as dimers under certain conditions.

The N-terminal amino acid sequence of plastocyanin from Stellaria media L. An exercise to establish criteria for the identification of residues from a sequenator.

The N-terminal amino acid sequence of plastocyanin from Stellaria media L. (chickweed) has been determined by means of an automatic sequencer. Amino acid derivatives obtained during the degradation were identified by a combination of t.l.c., g.l.c. and regeneration of the parent amino acid. The analytical results are presented for each sample, and the criteria used to establish the amino acid sequence are discussed.

The development of plastocyanin in greening bean leaves.

The plastocyanin content of etiolated bean leaves (Phaseolus vulgaris L.) was measured, and the development of the protein in response to light was followed. Measurements were made by quantitative extraction of plastocyanin and a sensitive assay with an O(2) electrode. The electron-paramagnetic-resonance (e.p.r.) signal of oxidized plastocyanin was used as an independent check on the validity of the assay method, and on the thoroughness of extraction. After an initial lag period, the amount of plastocyanin in greening bean leaves increased to reach a maximum after 50h illumination. The chlorophyll/plastocyanin ratio reached a maximum value of 200 irrespective of the light intensity at which greening was carried out, suggesting that the synthesis of the two components is co-ordinated. Experiments involving treatment of etiolated seedlings with brief periods of light of different spectral composition indicated that phytochrome is involved in plastocyanin synthesis. The lack of inhibition of plastocyanin synthesis by specific inhibitors of chloroplast protein synthesis suggests that the protein is synthesized on cytoplasmic ribosomes. The data are discussed in relation to the development of ferredoxin in greening bean leaves.

Quantitative studies on ferredoxin in greening bean leaves.

Two methods of measuring small amounts of the iron-sulphur protein ferredoxin are described. One involves measurements of the signal at g=1.96 produced by reduced ferredoxin in an e.p.r. (electron-paramagnetic-resonance) spectrometer; the other depends on the rate of ferredoxin-dependent electron transport in a chloroplast bioassay measured in an O(2) electrode. These methods of measurement were used to examine the development of ferredoxin during the greening of etiolated bean leaves. Ferredoxin is present in low concentrations in the leaves and cotyledons of 14-day-old etiolated beans (Phaseolus vulgaris L. var. Canadian Wonder), and develops in a linear manner with time when the leaves are illuminated. This synthesis appears to be independent of chlorophyll synthesis during the early stages of greening. However, the chlorophyll/ferredoxin ratio reaches a final value of approx. 360 irrespective of the light intensity, indicating the existence of a control mechanism operative in deciding the stoicheiometry of these components in the mature chloroplast. The ferredoxin synthesis appears to be light-dependent, and red light is the most effective in its promotion. The effect of red illumination is not reversed by far-red light, indicating the absence of a phytochrome control of ferredoxin synthesis. From experiments using specific inhibitors of chloroplast protein synthesis, it is concluded that ferredoxin is synthesized on cytoplasmic ribosomes.