A 3-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 fin al reduction of NADP(+) to NADPH. Herein, we report the engineering an d characterization of a novel protein molecule in which the electron-c arrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The ge ne 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 introduce d between the C-terminus of ferredoxin I and the N-terminus of the fla voenzyme. The chimera was purified to homogeneity and characterized. T he M-r of the chimera apoprotein was 45 800 as determined by mass spec trometry, in agreement with the expected value of 45 846, Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprote in. The chimera was found active as a diaphorase and, more interesting ly, highly efficient as a cytochrome c reductase, without need for fre e ferredoxin addition in the assay medium, Several lines of evidence i ndicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological c omplex. Thus, the fusion protein could be a useful tool for studying t he mechanism of protein-protein recognition and electron transfer in t he ferredoxin-ferredoxin-NADP(+) reductase system.