| 1. |
- Olloqui-Sariego, José Luis, et al.
(författare)
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Influence of tryptophan mutation on the direct electron transfer of immobilized tobacco peroxidase
- 2020
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 351
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Tidskriftsartikel (refereegranskat)abstract
- A major challenge in the design of electrochemical biodevices is to achieve fast rates of electron exchange between proteins and electrodes. In this work, we show that a significant increase in the direct electron transfer rate between a graphite electrode and Tobacco Peroxidase takes place when a surface exposed leucine, located in the vicinity of the heme pocket, is replaced by tryptophan. The analysis of the Fe(III)/Fe(II) voltammetric responses of native and mutated proteins, as a function of solution pH and temperature, leads to similar values of the reduction entropy and reorganization energy, but to a higher electronic coupling in the case of the mutant. In addition, the mutated and native proteins are shown to display similar electrocatalytic activities to reduce hydrogen peroxide at positive potentials, indicating that the molecular structure of the heme pocket is largely unaffected by the mutation.
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| 2. |
- Olloqui-Sariego, José Luis, et al.
(författare)
-
Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.
- 2015
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 87:21, s. 10807-10814
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Tidskriftsartikel (refereegranskat)abstract
- Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.
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| 3. |
- Olloqui-Sariego, José Luis, et al.
(författare)
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The Fe (III)/Fe(II) redox couple as a probe of immobilized tobacco peroxidase : Effect of the immobilization protocol
- 2019
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 299, s. 55-61
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Tidskriftsartikel (refereegranskat)abstract
- Non-turnover voltammetry is a sensitive tool to characterize the electrochemical properties of redox proteins. However, the catalytically competent oxidation states of most peroxidases do not display the required electrochemical reversibility. In this report, we circumvent this limitation and exploit the voltammetric response associated with the Fe(III)/Fe(II) redox couple of tobacco peroxidase to probe the energetics and electronic connectivity of the heme pocket. We have applied this approach to rationalize the previously reported influence of the immobilization protocol on the electrocatalytic activity of tobacco peroxidase. To decouple proton and electron transfer steps, measurements have been carried out over the 3 ≤ pH ≤ 9 range and a 1e−/2H+ ladder scheme has been adopted for their analysis. At each pH, thermodynamic and kinetic parameters associated with the Fe(III)/Fe(II) redox conversion were determined as a function of temperature in the 0-30 °C range. Reduction entropies and reorganization energies displayed different values for covalently immobilized and physisorbed enzymes, pointing to a larger involvement of the solvent in the last case. These findings, together with a larger electronic coupling between the prosthetic group and the electrode, are indicative of a partial denaturation of the physisorbed enzymes as the origin of their lower electrocatalytic activity.
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