Removal of ferredoxin:NADP+ oxidoreductase from thylakoid membranes, rebinding to depleted membranes, and identification of the binding site.

Ferredoxin-NADP+ oxidoreductase associates with thylakoid membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low ionic strength media. The nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid is uniquely able to remove the more tightly bound pool of enzyme, without solubilization of major membrane proteins. The reconstitution of reductase onto depleted thylakoid membranes requires available membrane binding sites and cations, in order of effectiveness trivalent greater than divalent greater than monovalent. The hetero/bifunctional 125I-iodinated Denny-Jaffe cross-linking reagent yields a 54-kDa, covalently cross-linked adduct between ferredoxin-NADP+ oxidoreductase and a component of the thylakoid membrane. Our results show that the more tightly bound pool of enzyme is associated with the 17.5-kDa reductase-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051).