AN INVESTIGATION OF THE RELATIONSHIP BETWEEN THE STRUCTURE AND FUNCTION OF THE BLUE COPPER ELECTRON TRANSPORT PROTEIN PLASTOCYANIN USING THIN-LAYER, STEADY-STATE SPECTROELECTROCHEMISTRY

A method is described for generating steady-state currents between two closely-spaced, coplanar electrodes in a thin solution layer and simultaneously monitoring the absorbance of the electroactive species. Semiempirical equations are presented which quantitatively explain data from steady-state electrochemical and spectroelectrochemical experiments on model redox couples and on the electron transport protein plastocyanin.;Plastocyanin carboxyl groups are converted into positively charged amide groups by reaction with ethylenediamine (EDA) in the presence of a water soluble carbodiimide. Singly modified plastocyanin exhibited at least a 17 mV positive shift in formal potential. This positive shift cannot be explained in terms of a simple alteration in the electric field experienced by the copper ion upon replacing a negatively charged carboxyl group a known distance away with a positively charged EDA group.;The formal potential of native plastocyanin shifts in a positive direction upon lowering the pH. However, the formal potential of plastocyanin with its east face protonation sites blocked by EDA modification is unaffected by lowering the pH. This suggests a cooperative relationship between the east face residues and the copper center in which the copper center is sensitive to the extent of protonation on the east face. This concept is supported by analogous kinetic experiments in which the rate of oxidation of plastocyanin maximally modified with EDA was much less sensitive to lowering the pH than that observed with unmodified plastocyanin. Plastocyanin with a copper center sensitive to the extent of protonation on the east face, may play a role in the regulation of photosynthetic electron transport.;The reduced diffusion coefficient for plastocyanin was determined using steady-state electrochemistry (8.7 x 10('-7) cm('2) sec('-1)). Formal potentials for plastocyanin 372 mV (+OR-) 5 mV vs. NHE) were determined using either single-electrode or twin-electrode spectroelectrochemistry using 1,1'-bis(hydroxymethyl)ferrocene as a redox mediator. The formal potential of plastocyanin was independent of mediator concentration within at least a 10-fold concentration range of the mediator. The formal potential was used as a structure-sensitive probe in studies of the protein under various experimental conditions.