Plastocyanin conformation: the effect of the oxidation state on the pKa of nitrotyrosine-83.

Plastocyanin treated with tetranitromethane was nitrated at a single location, Tyr-83. Tyr-83 and its neighboring negative charges have been implicated as a binding site for positively charged redox agents (Chapman, S.K., Watson, A.D. and Sykes, A.G. (1983) J. Chem. Soc. Dalton Trans. 1983, 2543-2548). No effect was observed on either the plastocyanin midpoint redox potential or its reaction kinetics with P-700+ and cytochrome f. This makes nitration an ideal spectroscopic probe for monitoring changes in the environment of Tyr-83. The pKa of the nitrotyrosine was 8.6 and 8.3 for reduced and oxidized plastocyanin, respectively, indicating that the charge on the copper atom is 'felt' at Tyr-83. The high pKa value for both forms indicates that Tyr-83 is in a negatively charged environment, near residues Nos. 42-45 and Nos. 59-61. The extinction of the nitrotyrosine chromophore at 360 nm was not affected by a change in redox state. However, the ellipticity of this transition was greater for the oxidized form, indicating that environment of Tyr-83 is dependent upon the charge on the copper atom. This suggests an electrostatically driven conformational change at Tyr-83. A conformational change at Tyr-83 could regulate the binding of plastocyanin with its reaction partners in order to promote smooth electron transport.