Ferredoxin:NADP+ Oxidoreductase Is a Subunit of the Chloroplast Cytochrome bfComplex

Abstract

Purified detergent-soluble cytochromeb₆f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300–350 electrons percyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f α-band and the Qy chlorophylla 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 ∼ 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of ∼0.9 (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-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplastb₆f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b₆ subunit. It was inferred that the b₆f 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.