Plastocyanin conformation: The effect of the oxidation state on the p Ka of nitrotyrosine-83

Abstract

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 p K a 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 p K a 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.