Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex

Purified detergent-soluble cytochrome b(6)f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha -band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M-r similar to 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of similar to0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP(+) oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinolferricyanide activity. The activity of the FNR in the chloroplast b(6)f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b(6) subunit. It was inferred that the b(6)f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.