Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with inorganic redox partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+

Plastocyanin (PCu) from spinach leaves has been singly NO2-modified, purified by FPLC, and the position of modification at Tyr83 confirmed by trypsin digestion and amino-acid sequencing. Electron-transfer reactions of native and NO2-modified PCu with the inorganic redox partners Fe(CN)6]3- and Co(phen)3]3+, as oxidants for PCu(I), and Fe(CN)6]4- and Co(phen)3]2+ as reductants for PCu(II), have been studied as a function of pH. The acid dissociation constant for the phenolic group on NO2-Tyr83 PCu is 8.78 (average) for reduced, and 8.10 for oxidised protein, as compared to values greater than 10 for native protein. At I = 0.10 M (NaCl) NO2-modification brings about a 20 mV increase in reduction potential at pH less than 7 and deprotonation of the phenolic group a 20-25 mV decrease, both transmitted to and effective at the active site. Deprotonation brings about a 48% increase in rate for Fe(CN)6]3- and a 47% decrease for Fe(CN)6]4- in accordance with these changes. In the case of Co(phen)3]3+, which reacts substantially at the remote site in the vicinity of Tyr83, the influence of deprotonation on the active site is supplemented by the negative charge of the phenolate, and a total increase of 131% is observed. These results can be understood on the basis of the electron-transfer theory, and add support to the belief that electron transfer kinetics of negatively and positively charged reactants are dominated by different sites on PCu for electron transfer, namely adjacent (close to His87) and remote (close to Tyr83), respectively.