Electrochemical characterization of dehaloperoxidase adsorbates on COOH/OH mixed self-assembled monolayers

https://doi.org/10.1016/j.jelechem.2013.05.023Get rights and content

Highlights

  • Dehaloperoxidase adsorption takes place on COOH/OH self-assembled monolayers.

  • Voltammetrically determined surface concentration is time dependent.

  • Dynamic reorientation of latent adsorbates is consistent with voltammetry.

  • A model based on distributions of orientation and electroactivity is proposed.

  • Dehaloperoxidase structure and surface properties are compatible with the model.

Abstract

Electrochemical characterization of Amphitrite ornata dehaloperoxidase, a monomeric hemoglobin, adsorbed on COOH/OH-terminated alkanethiolate mixed self-assembled monolayers (SAMs) is reported. Adsorption was achieved by simple electrode exposure to low ionic strength protein solution at pH 6.0, and cyclic voltammetry (CV) was employed to measure surface concentration, electron transfer rate, and formal potential under anaerobic conditions. Surface concentration values determined by CV were found to be inversely proportional to scan rate, a behavior that is different than that observed for electron transfer proteins such as cytochrome c. We attribute this behavior to a dynamic heterogeneous adsorbate layer that permits some fraction of the molecules to undergo reorientation during a CV scan. A model was proposed that classifies adsorbates among three populations with respect to their most stable orientations, namely, electroactive adsorbates, latent electroinactive adsorbates capable of undergoing reorientation to electroactive states, and electroinactive adsorbates. Electrochemical results and the proposed model are discussed in terms of the tertiary structure and surface properties of dehaloperoxidase and are compared to the docking and electron transfer reactions of the myoglobin/cytochrome b5 system. Examination of the effects of ionic strength and mixed SAM composition provide support for the proposed model.

Introduction

Orientation specificity of protein binding is a fundamental property that controls numerous biochemical reactions such as respiration and signal transduction [1], [2], [3]. On electrode surfaces, immobilized enzyme orientation is a critical factor for the successful operation of biosensors based on direct electron transfer (“third generation biosensors”) [4], [5]. Interfacial chemistry between proteins and their binding partners is thus of great interest and significance. Formation of functionalized alkanethiolate self-assembled monolayers (SAMs) represents a versatile means for tailoring the chemical composition of electrodes and other types of surfaces [6], [7], including biomimetic surfaces designed to study protein chemistry [8], [9], [10], [11], [12]. We present here an electrochemical characterization of dehaloperoxidase (DHP) adsorbed on COOH/OH-terminated alkanethiolate mixed SAM surfaces. DHP, a coelomic hemoglobin from the marine annelid Amphitrite ornata, is the first globin discovered to have a biologically relevant peroxidase function [13], [14], [15]. In a recent publication [16], we reported initial voltammetric results that showed that DHP can be adsorbed in a chemically reversible manner on COOH/OH mixed SAMs although the stability of the voltammetric response was not robust. Evidence was also presented that revealed the dynamic nature of the adsorbed layer of protein. In the present report, we first describe an improved purification procedure that has dramatically enhanced the voltammetric stability of adsorbed DHP. Subsequently, results of cyclic voltammetry (CV) studies will be presented with a focus on developing a better understanding of the nature of interfacial interactions that occur between DHP and the electrode surface. To this end, effects of ionic strength (μ) and SAM composition on the voltammetry of adsorbed DHP were investigated.

The concept of a protein–protein electron transfer (ET) complex can serve as a useful paradigm for adsorbed proteins on electrodes (i.e., protein–electrode complexes). Rates of electron transfer within such complexes are highly dependent on the properties of the surfaces involved and the interactions that result. The simplest model (“simple docking”) involves a single energy minimum corresponding to a protein–protein complex wherein the thermodynamically most stable binding configuration and the configuration that results in optimal ET coincide [17]. Alternatively, a “gated” ET model describes protein–protein complexes wherein the most stable binding configuration differs from the most reactive ET configuration. Consequently, the rate of ET can become “gated” by the rate of interconversion between the configurations. For example, it has been proposed that cytochrome c (Cyt c) ET becomes “gated” when COOH SAM-modified gold electrodes are thin enough such that the intrinsic rate of ET exceeds the rate of configurational interconversion [18]. More recently, a new model has been proposed by Hoffman’s group to account for bimolecular rates of ET between myoglobin (Mb) and cytochrome b5 (Cyt b5). Their model, referred to as “dynamic docking”, postulates the existence of a broad distribution of weakly bound protein–protein configurations, of which only a small fraction are actually capable of ET. Furthermore, the ET-capable configurations, which involve protein orientations that engender heme-edge interactions between the proteins, are not the most stable binding configurations [1], [17], [19]. Accordingly, binding thermodynamics and ET kinetics can effectively become decoupled as shown by Hoffman’s group in a series of elegant experiments using horse Mb and Cyt b5 [1], [17], [19], [20].

The interactions between DHP and COOH/OH SAMs bear some resemblance to those between Mb and Cyt b5, which have been characterized as primarily electrostatic [1], [17], [19], [20]. DHP is very similar to Mb with regard to tertiary structure, overall surface charge (nearly neutral), and local charge distribution (slightly positive) near its heme-edge region where ET takes place [1], [13], [15]. Cyt b5, on the other hand, is an anionic protein and features a negatively charged heme edge region. The C8OH/C7COOH mixed SAMs that we used in this study exhibit pK1/2 values in the range of 6–6.5 [21]. Thus, the SAM surface will be partially deprotonated at the pH 6.0 condition used to acquire the results reported here, and the SAM surface will carry a negative charge, as does Cyt b5. Although the heterogeneous planar surface of a SAM presents a distinctly different interfacial environment than does the protein surface of Cyt b5, which has been characterized as “football-shaped” [17], we will attempt to draw parallels where possible. This work further reaffirms a role for self-assembled monolayers in mimicking biological interfaces and the notion of protein-electrode complexes for investigating biochemical phenomena.

Section snippets

Materials and methods

Gold electrodes (100 nm Au/50 Å Ti/glass) were purchased from Evaporated Metal Films (Ithaca, NY). Alkanethiols were purchased from Dojindo Molecular Technologies, Inc., and gold powder (<45 μm) was purchased from Sigma–Aldrich. All other chemicals were purchased from Sigma–Aldrich or VWR International and were used as received. Water was deionized and further purified using a Barnstead purification system (17.3  cm upon delivery).

DHP refers to the His-tag form of the DHP A isoenzyme, with the

Stability of voltammetric responses

Previously reported voltammetry of adsorbed DHP on COOH/OH mixed-SAM gold electrodes suffered from temporal instability with the faradaic current typically diminishing to a minor fraction of its original value within ∼1 h of replacing the DHP adsorption solution with low ionic strength buffer [16]. Fig. 1a, however, demonstrates that much improved voltammetric stability can be obtained by exposing chromatographically purified DHP samples to a COOH/OH SAM-modified gold powder (AuP) before

Conclusions

Using cyclic voltammetry, we have investigated the electrochemical properties of a monomeric hemoglobin, dehaloperoxidase (DHP), adsorbed on COOH/OH-terminated alkanethiolate mixed-SAMs. In light of DHP’s tertiary structure and surface properties, which typify common globins, insights gleaned from the present work are germane to other myoglobin and hemoglobin voltammetry. Scan rate, ionic strength, and mixed-SAM composition results point to an adsorbate layer that is very heterogeneous with

Acknowledgements

This work was supported in part by Army Research Office Grant 51432-CH-SR. We are indebted to Professor Reza A. Ghiladi for allowing us the use of his glove box.

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    Present address: SRI International, Harrisonburg, VA 22802, United States.

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