A Systematic Study of the Influence of Peptide Modification of a Gold Electrode on the Cyclic Voltammetry of Pseudoazurin from Alcaligenes faecalis Strain S‐6

Abstract

The influence of peptide-protein interactions on the electrochemistry of copper-containing pseudoazurin from Alcaligenes faecalis strain S-6 has been investigated by covalently binding cysteine-containing hexapeptides to a gold electrode surface. The hexapeptides contain three cysteines in the same positions with the remaining amino acids varied to give mixed charge (lysine, threonine, alanine), positive (lysine), overall neutral (alanine), and negative (glutamate) chemically modified electrode surfaces. These systematic variations in the amino acid sequence lead to large variations in voltammetric behavior for the Cu(II) ! Cu(I) heterogeneous pseudoazurin redox process encompassing fully reversible and diffusional, transitionally adsorbed, or strongly adsorbed forms of voltammetry. The variations in voltammetric behavior may be related to electrostatic interactions between the charges from the hexapeptide electrode modifiers and surface charges of pseudoazurin. A possible description of the pseudoazurinelectrode surface interaction is given.

Orientational Control over Nitrite Reductase on Modified Gold Electrode and Its Effects on the Interfacial Electron Transfer

“…The enzyme electrodes with SAMs of pure 8-OH, 8-NH 2 , 7-CH 3 , and 7-COOH, as well as mixtures of 8-OH/7-COOH and 8-OH/7-CH 3 did not show catalytic waves after NO 2 – addition, despite the fact that the protein is able to physisorb on all these electrodes ( vide infra ). This indicates that the L93C NiR orientation on the surface is key for its activity, in line with previous studies. , Figure A (inset, black solid line) shows a typical voltammetric response of L93C NiR adsorbed on a 20%:80% 8-NH 2 :8-OH SAM electrode, recorded in the absence of nitrite. Besides the redox peaks due to the L93C NiR oxidation and reduction, a slight catalytic wave can be observed, which we suggest is due to a relatively slow reduction of oxygen to hydrogen peroxide by the enzyme, as previously observed .…”

“…This indicates that the L93C NiR orientation on the surface is key for its activity, in line with previous studies. 7,8 Figure 1A (inset, black solid line) shows a typical voltammetric response of L93C NiR adsorbed on a 20%:80% 8-NH 2 :8-OH SAM electrode, recorded in the absence of nitrite. Besides the redox peaks due to the L93C NiR oxidation and reduction, a slight catalytic wave can be observed, which we suggest is due to a relatively slow reduction of oxygen to hydrogen peroxide by the enzyme, as previously observed.…”

“…Nitrite reductase (NiR) from Alcaligenes faecalis S-6 is a copper-containing enzyme (Scheme ), which takes part in microbial denitrification processes by catalyzing the one electron reduction of nitrite to nitric oxide: NO 2 – + 2H + + e – = NO + H 2 O. , The enzyme is a homotrimer, in which each subunit contains a type-1 Cu site and a type-2 Cu site. , The type-1 copper site, located close to the protein’s surface, accepts electrons coming from the enzyme’s natural donor, pseudoazurin, and acts as an electron relay by which electrons are transferred to the type-2 copper site, buried deep inside the enzyme. ,− The latter site, together with a water network around an aspartate and a histidine residue forms the catalytically active site of the enzyme. ,,, Previously, it was shown that the catalytic cycle of NiR operates via a random sequential mechanism (RSM). ,, Spectroelectrochemistry studies, in which a novel fluorescent technique , was used to examine the oxidation state of the type-1 Cu center during enzymatic turnover of NiR immobilized on modified gold, revealed that interfacial electron transfer (ET) between electrode and enzyme is heterogeneous and constitutes the rate-limiting step for catalysis by the immobilized NiR. Earlier spectroelectrochemical studies of fluorescently labeled azurin immoblized on SAM-covered gold electrodes had already provided proof for a considerable spread in ET rates and midpoint potential of the immobilized blue copper protein .…”

Spectroelectrochemical Investigation of Intramolecular and Interfacial Electron-Transfer Rates Reveals Differences Between Nitrite Reductase at Rest and During Turnover

“…Copper-containing nitrite reductase (NiR) catalyzes an one-electron reduction of nitrite to nitric oxide: NO 2 – + 2H + + e – → NO + H 2 O in denitrifying organisms . NiR is a homotrimer, in which each subunit contains a type-1 and a type-2 copper site (Figure ). , The type-1 Cu site, which is located close to the protein’s surface, accepts electrons from its natural donor, pseudoazurin, and transfers them to the buried type-2 Cu site where nitrite is reduced. ,− In the type-1 copper center of green NiR from Alcaligenes faecalis, the copper atom is coordinated by two histidines, one methionine, and one cysteine . A ligand-to-metal charge transfer (LMCT) from the sulfur (cysteine) to the oxidized copper (Cu 2+ ) gives rise to absorption bands around 450 and 590 nm, which disappear when the copper gets reduced (Cu 1+ ).…”

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

“…Several works have reported the direct electrochemistry of blue copper proteins, including PAz from Achromobacter cycloclastes [21,22] and from Alcaligenes faecalis [23]. Few works dealing with the electrochemistry of other peroxidases have been published, with monohaem CCP being the most studied [24,25].…”