Electrochemical Investigations of Poplar, Spinach, Cucumber, and Parsley Plastocyanins at Conventional and Microsized Carbon Electrodes

Thermodynamic and kinetic aspects of the electrochemistry of the ‬blue‭ copper protein plastocyanin from poplar, spinach, cucumber, and parsley have been studied at both macro- and microsized carbon-disk electrodes. Reversible formal potentials E_F⁰ at 3 °C have been determined by cyclic voltammetry in the range 3.6 ⩽ pH ⩽ 7.6. Above pH 6.6 the reversible potentials of all four plastocyanins are independent of pH and have limiting values of 389 ± 7 mV vs NHE. Below pH 6.6 the reversible potentials are functions of pH. Poplar, spinach, and cucumber plastocyanins have similar pH dependences, but the curve for parsley plastocyanin is shifted by ~0.7 pH unit toward higher pH. While the reasons for this difference and for some other details of the pH dependences are not completely understood, the fact that parsley plastocyanin behaves differently from the others is consistent with antecedent observations of redox kinetics. The electrochemical kinetic behavior of the plastocyanin/graphite interface is complex and is influenced by a competing protein adsorption process. Under the experimental conditions employed, only a small fraction (< 10%) of the electrode surface is active so that the conventional linear diffusion model (in which the entire surface participates) is untenable. Instead, the apparent kinetic parameters (peak separations) of the voltammetric data recorded with normal macrosized electrodes are strongly influenced by nonlinear mass transport to micron- to submicron-sized active sites. When nonlinear diffusion is taken into account, the experiments are consistent with very fast electron transfer between plastocyanin and the active sites of the carbon electrodes. The detection of nonlinear diffusion, combined with the detection of a voltammetric response for free Cu²⁺, provides a sensitive probe of the integrity of the protein; the voltammogram of the intact protein is modified in the presence of apoprotein or denatured protein. The data obtained from measurements at conventionally sized electrodes are confirmed by results obtained at carbon microelectrodes. Not only is radial diffusion found to be the predominant mechanism of mass transport (as expected from the small size of the electrodes) but also partial blocking of even these electrodes can be clearly demonstrated.