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.