Voltammetry of Plastocyanin at a Graphite Electrode:  Effects of Structure, Charge, and Electrolyte

Abstract

Comparative voltammetric studies on Anabaena variabilis plastocyanin (positively charged at neutral pH) and spinach and poplar plastocyanins (negatively charged at neutral pH) have been undertaken at an edge-plane graphite electrode as a function of ionic strength, pH, and Mg(2+) concentration at 3 degrees C. The aim was to provide a more detailed understanding of the influence of the electrode-protein (solution) interfacial characteristics, as well as the variation of the formal potential with both the nature of the plastocyanin species and the pH. As might be expected, some of the interfacial properties associated with the positive charge on A. variabilis plastocyanin are the opposite of those observed with the negatively charged plastocyanins. For example, the linear diffusion component of the mass transport process for A. variabilis plastocyanin under the conditions of cyclic voltammetry is decreased and the radial diffusion component is increased by the addition of Mg(2+), whereas the reverse occurs with poplar and spinach plastocyanins. The voltammetrically determined reversible potentials for A. variabilis plastocyanin are considerably less positive than those for spinach and poplar plastocyanins, in agreement with values calculated from chemically based redox studies. Ionic strength effects, as determined by addition of NaClO(4) over the concentration range 0.005-0.20 M, are negligible for all three proteins. The addition of Mg(2+) causes a significant shift in the reversible potential toward more positive values for spinach and poplar plastocyanin but only a small positive shift for A. variabilis plastocyanin. The difference is attributed to a specific binding effect. The addition of Mg(2+) also dramatically alters the pH dependence of the reversible potential, indicating that the equilibrium between the protonated and unprotonated forms of reduced plastocyanin is modified by binding of Mg(2+) to the protein. It is concluded that the effects of biologically relevant redox-inactive cations such as Mg(2+) or Ca(2+) have to be considered carefully in studies of the redox chemistry of metalloproteins.

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

“…This behavior indicates that Cu 2 + reduction is coupled to a protonation process at the active site that involves the solvent-exposed Cu-binding His residue, which detaches from the metal. [9,15,17,23,28] The low-pH region of the E8'/pH profiles was fitted to the following single acid-base equilibrium equation, which applies to the above conditions: [17,23,29] The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(i) ion of the His87 ligand have been evaluated.…”

“…The consequent dramatic increase in the reduction potential of the copper center disables the protein functionally. [7,9,15] Therefore, the acid transition, which is fully reversible, has been proposed to play a physiological role as a molecular redox switch. [7,9,16] The pK a value of the acid transition in cupredoxins is remarkably species-dependent, and follows the order: amicyanin @ plastocyanins !…”

Ligand Loop Effects on the Free Energy Change of Redox and pH-Dependent Equilibria in Cupredoxins Probed on Amicyanin Variants

“…The latter is a key parameter for protein function ( − ). It can be dramatically affected by pH-dependent conformational transitions, which may bear on the electron donor and acceptor capability of the protein ( − ). Such a pH-induced transition, thus, may act as a molecular switch of redox activity.…”

Thermodynamics of the Acid Transition in Blue Copper Proteins

“…Copper and iron proteins serving as biological electron carriers are known to undergo pH-induced conformational transitions involving changes in the first coordination sphere of the metal. In particular, the so-called alkaline transition in oxidized class I cytochromes c and the acid transition in reduced blue copper proteins have been thoroughly investigated by spectroscopic and structural means ( − ). For cytochromes c , additional low-spin Fe(III) conformers are known to form when the pH is raised above 8, in which the native axial methionine ligand is replaced by a surface lysine, the ligand exchange being triggered by deprotonation of an as yet unidentified residue ( , ).…”

“…In both cases, ligand replacement/detachment from the metal causes a dramatic change in the reduction potential, responsible for the redox inactivation of the protein, in the sense that it becomes unable to carry out its physiological function. For that reason, hypotheses have been put forward on the possible physiological role of these transitions (which are both reversible) as biological redox switches ( , ).…”