The complex formed between plastocyanin and cytochrome c. Investigation by NMR spectroscopy.
Spinach plastocyanin and horse heart cytochrome c have been shown, by monitoring the behaviour of the hyperfine-shifted heme resonances of Fe(III) cytochrome c on titration with Cu(II) plastocyanin, to form a 1:1 complex with a dissociation constant of 67 mM (D2O, pH* 7.0, 300 K). The interaction sites on the plastocyanin surface have been investigated in one- and two-dimensional NMR experiments involving competition for plastocyanin between cytochrome c and the paramagnetic cation Cr(NH3)(3+)6. The plastocyanin resonances which are paramagnetically broadened in the spectrum of plastocyanin alone are also broadened in the spectrum of the mixture of the two proteins. This shows that, on the NMR time scale, no plastocyanin residues are hidden from Cr(NH3)(3+)6 by complexation with cytochrome c. [It has been shown that Cr(NH3)(3+)6 does not disrupt formation of the complex between the two proteins.] It appears that initial complexation of cytochrome c takes place at the acidic east site of plastocyanin, and that the extensive negative electrostatic surface of plastocyanin accommodates the paramagnetic probe and cytochrome c simultaneously in a dynamic ternary complex. The location of the electron transfer site on plastocyanin is discussed.
Bagby
S
S
Inorganic Chemistry Laboratory, Oxford, England.
Driscoll
P C
PC
Goodall
K G
KG
Redfield
C
C
Hill
H A
HA
D000818:Animals; D001665:Binding Sites; D003300:Copper / Q000032:analysis; D003574:Cytochrome c Group / Q000032:analysis; D005290:Ferric Compounds / Q000032:analysis; D005296:Ferrous Compounds / Q000032:analysis; D006418:Heme / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000032:analysis; D010970:Plastocyanin / Q000032:analysis; D013499:Surface Properties
D003574:Cytochrome c Group; D005290:Ferric Compounds; D005296:Ferrous Compounds; D010940:Plant Proteins; D006418:Heme; D003300:Copper; D010970:Plastocyanin