The role of acidic residues of plastocyanin in its interaction with cytochrome ƒ

The role of the acidic patches of spinach plastocyanin in the interaction with a soluble form of turnip cytochrome ƒ was studied by a combination of site-directed mutagenesis, NMR spectroscopy and kinetic analysis. The charge of the two 'eastern' patches, consisting of conserved acidic residues 42-45 and 59-61 respectively, was altered by incorporation of neutral or positively charged groups. Up to four negative charges were deleted in six different mutants and a further mutant, Q88E, provided an additional negative charge in the same region. Overall second-order rate constants (k₂) for reduction by cytochrome ƒ were determined by stopped-flow spectrophotometry. A 2- to 3-fold decrease in k₂ was observed for each negative charge abolished, regardless of its position, and in Q88E there was a 20% increase. From the ionic strength dependence similar values for k₂ at infinite ionic strength were predicted for the native and mutant proteins, while the electrostatic attraction term decreased with each negative charge removed. The equilibrium constant for association (K_A) was determined from the change in T₂ of ¹H resonances of plastocyanin. Loss of negative charges caused marked decreases in K_A roughly in parallel with the decreases in k₂, which suggests that the main effect was on binding rather than the rate of intracomplex electron transfer. Taken together, these results provide convincing evidence for participation of residues of both acidic patches in the interaction with cytochrome ƒ.