The complex formed between plastocyanin and cytochrome c

Abstract

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.

Effects of Temperature on the Kinetics of the Gated Electron-Transfer Reaction between Zinc Cytochrome c and Plastocyanin. Analysis of Configurational Fluctuation of the Diprotein Complex

“…35, No. 47, 1996 15101 Zncyt/pc the positive patch around the exposed heme edge in cytochrome c interacts with the broad, negative patch in plastocyanin (King et al, 1985;Bagby et al, 1990;Roberts et al, 1991;Geren et al, 1983;Zhou et al, 1992). This study confirms the results of previous studies of the effects of ionic strength on the forward reaction (eqs 12 and 13) at a single temperature (Zhou & Kostic ´, 1991a, 1993b and adds to them an analysis of temperature effects and of activation parameters.…”

The identification of cation‐binding domains on the surface of microsomal cytochrome b5 using 1H‐NMR paramagnetic difference spectroscopy

“…In addition the ternary complex between cytochromes b5 and c detected by NMR methods may result from an association of two cytochrome c molecules close to this large domain [20, 221. In an analogous 'H-NMR study the surface of the type I blue copper protein plastocyanin remained accessible to the relaxation probe hexaamminechromium(III), Cr(NH3)2 ', in a 1 : 1 complex with ferricytochrome c [32]. Plastocyanin and cytochrome c are non-physiological redox partners forming a protein complex whose association constant is low ( K , = z 15 M-I, I = [32]; K, = < 150 M-', I = 0.1 M [50]). This may explain why these authors failed to detect any shielding in the plastocyanin/cytochrome c complex.…”

Effects of Mutations in Plastocyanin on the Kinetics of the Protein Rearrangement Gating the Electron-Transfer Reaction with Zinc Cytochrome c. Analysis of the Rearrangement Pathway

“…Because zinc cytochrome c and the wild-type cupriplastocyanin bear respective net charges of +6 and -8 at pH 7.0, and because they contain oppositely charged surface patches, these two proteins associate in solution at low ionic strengths. Much evidence shows that in the cyt/pc complexes the basic (positive) patch around the exposed heme edge abuts the broad acidic (negative) patch in plastocyanin (King et al, 1985;Bagby et al, 1990;Roberts et al, 1991;Zhou et al, 1992).…”