A three-domain iron-sulfur flavoprotein obtained through gene fusion of ferredoxin and ferredoxin-NADP+ reductase from spinach leaves.
Ferredoxin and ferredoxin-NADP+ reductase are the two last partners of the photosynthetic electron-transfer chain, responsible for the final reduction of NADP+ to NADPH. Herein, we report the engineering and characterization of a novel protein molecule in which the electron-carrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The gene was obtained by joining the cDNAs encoding the respective proteins and subsequently by deleting the intervening sequence between them by site-directed mutagenesis. No extra amino acid residues were introduced between the C-terminus of ferredoxin I and the N-terminus of the flavoenzyme. The chimera was purified to homogeneity and characterized. The M(r) of the chimera apoprotein was 45,800 as determined by mass spectrometry, in agreement with the expected value of 45,846. Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprotein. The chimera was found active as a diaphorase and, more interestingly, highly efficient as a cytochrome c reductase, without need for free ferredoxin addition in the assay medium. Several lines of evidence indicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological complex. Thus, the fusion protein could be a useful tool for studying the mechanism of protein-protein recognition and electron transfer in the ferredoxin-ferredoxin-NADP+ reductase system.
Aliverti
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Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Zanetti
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D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017343:Genes, Plant; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000378:metabolism; D010788:Photosynthesis; D018515:Plant Leaves; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D018724:Spinacia oleracea
D005288:Ferredoxins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D005287:Ferredoxin-NADP Reductase; D009245:NADH Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase