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

We study, by flash kinetic spectrophotometry on the microsecond time s cale, the effects of ionic strength and viscosity on the kinetics of o xidative quenching of the triplet state of zinc cytochrome c ((3)Zncyt ) by the wild-type form and the following nine mutants of cupriplastoc yanin: Leu12Glu, Leu12Asn, Phe35Tyr, Gln88Glu, Tyr83Phe, Tyr83His, Asp 42Asn, Glu43Asn, and the double mutant Glu59Lys/Glu60Gln. The unimolec ular rate constants for the quenching reactions within the persistent diprotein complex, which predominates at low ionic strengths, and with in the transient diprotein complex, which is involved at higher ionic strengths, are equal irrespective of the mutation. Evidently, the two complexes are the same. In both reactions, the rate-limiting step is r earrangement of the diprotein complex from a configuration optimal for docking to the one optimal for the subsequent electron-transfer step, which is fast. We investigate the effects of plastocyanin mutations o n this rearrangement, which gates the overall electron-transfer reacti on. Conversion of the carboxylate anions into amide groups in the lowe r acidic cluster (residues 42 and 43), replacement of Tyr83 with other aromatic residues, and mutations in the hydrophobic patch in plastocy anin do not significantly affect the rearrangement. Conversion of a pa ir of carboxylate anions into a cationic and a neutral residue in the upper acidic cluster (residues 59 and 60) impedes the rearrangement. C reation of an anion at position 88, between the upper acidic cluster a nd the hydrophobic patch, facilitates the rearrangement. The rate cons tant for the rearrangement smoothly decreases as the solution viscosit y increases, irrespective of the mutation. Fittings of this dependence to the modified Kramers's equation and to an empirical equation show that zinc cytochrome c follows the same trajectory on the surfaces of all the plastocyanin mutants but that the obstacles along the way vary as mutations alter the electrostatic potential. Mutations that affect protein association (i.e., change the binding constant) do not necess arily affect the reaction between the associated proteins (i.e., the r ate constant) and vice versa. All of the kinetic and thermodynamic eff ects and noneffects of mutations consistently indicate that in the pro tein rearrangement the basic patch of zinc cytochrome c moves from a p osition between the two acidic clusters to a position at or near the u pper acidic cluster.