Protein-film voltammetry

The large size of redox enzymes means that diffusion to an electrode surface will be prohibitively slow, and, for enzyme in solution, an electrochemical response is usually only observed if small, soluble electron transfer mediator molecules are added. In this chapter, discussion is limited to examples in which the enzyme of interest is attached to the electrode surface. Electrochemical experiments on enzymes can be very simple, involving direct adsorption of the protein onto a carbon or modified metal surface from dilute solution. Protein film voltammetry, a method in which a film of enzyme in direct... 

Barker CD, Reda T, Hirst J. 2007. The flavoprotein suhcomplex of complex I (NADH ubiquinone oxidoreductase) from bovine heart mitochondria Insights into the mechanisms of NADH oxidation and NAD reduction from protein film voltammetry. Biochemistry 46 3454-3464. 

Heering HA, Wiertz FGM, Dekker C, de Vries S. 2004. Direct immobilization of native yeast Iso-1 cytochrome c on bare gold Fast electron relay to redox enzymes and zeptomole protein-film voltammetry. J Am Chem Soc 126 11103-11112. 

Leger C, Elliott SJ, Hoke KR, Jeuken LJC, Jones AK, Armstrong FA. 2003. Enzyme electrokinetics Using protein film voltammetry to investigate redox enzymes and their mechanisms. Biochemistry 42 8653-8662. 

In the last 20 years, a big effort has been made to characterize different reaction schemes taking place at modified electrodes, with special focus on the case of biomolecules in what has been called Protein Film Voltammetry [79-83]. Among the different situations analyzed with multipulse techniques (including Cyclic Voltammetry), it can be cited the surface ECE process [89, 90] and surface reactions preceded by homogeneous chemical reactions [91]. For a more detailed revision of the different mechanisms analyzed in the case of SWV, see [19]. 

Important advantages are gained if proteins are immobilized at the electrode surface, hence giving an electroactive film that is ideally of monolayer coverage. This approach has been termed protein film voltammetry and one of its major advantages is that microscopic quantities of the protein are required and thermodynamic and kinetic information can be obtained with higher accuracy and resolution. 

Armstrong, F. A., Heering, H. A., and Hirst, J., 1997, Reactions of complex metalloproteins studied by protein-film voltammetry, Chem. Soc. Rev. 26 169nl79. 

Interfacial Electron Tranter in Molecular and Protein Film Voltammetry 87... 

Figure 2.6 High-resolution in situ STM images of some pure and functionalized alkanethiols and protein film voltammetry linkers on Au(lll) electrode surfaces in various aqueous buffers, (a) Bare reconstructed Au(lll) surface ...

Armstrong, F.A. (2002) Insights from protein film voltammetry into mechanisms of complex biological electron-transfer reactions. Journal of the Chemical Society. Dalton Transactions, 551-571. 

Anderson, L.J., Richardson, D.J., and Butt, J.N. (2001) Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity. Biochemistry, 40, 11294-11307. 

Figure 4-1. Protein film voltammetry as a technique for studying redox enzyme mechanisms. The catalytic current-potential profile provides information on the rate-defining catalytic processes occurring within the enzyme. It is important that interfacial electron transfer is facile and information is not masked by limitations due to tlie transport of substrate and product for this reason the rotating disc electrode is an important tool in these studies.

As a tool for mechanistic enzymology, protein film voltammetry benefits... 

Figure 4-2. Cartoons of the ways in which proteins are attached to electrodes for protein film voltammetry. (A) Pyrolytic graphite edge (PGE) electrode. (B) SAM modified metal electrode. (C) Layer-by-layer polyion adsorption. (D) Electrode confined bilayers.

Two of the electrochemical techniques used in protein film voltammetry are shown in Fig. 4-3. In cyclic voltammetry the electrode potential is swept in a linear manner back and forth between two limits. The rate at which the potential is scanned defines the time scale of the experiment and this can be varied from < 1 mV s to > 1000 V s . This is a very large dynamic range, and it is possible to carry out both steady-state and transient experiments on the same sample of enzyme. " Cyclic voltammetry is important because it provides the big picture and produces a signal that links the reaction or active site of interest to a particular potential. In chronoamperometry, the current is monitored at a constant potential following a perturbation such as a step to this potential or addition of a substrate. This experiment is important because it separates the potential and time dependencies of a response. In both types of experiment, it is usually important to be able to rotate the electrode in order to control transport of the substrate and product to and from the enzyme film. 

Anderson, L.J., Protein film voltammetry and spectroscopic studies ofibacterial nitrate reductases, Ph.D. Thesis, University of East Anglia, Norwich, U.K, 2002. 

These observations apply to protein film voltammetry as well but other observations are here important. A physical effect of some importance is that the protein/film interaction appears to depend on the film thickness. Some distance seems to be needed before this soft matter interaction relaxes into a stable pattern. This is seen for example by the biphasic distance dependence... 

Figure 8-11. Overview of metalloproteins recently characterized by single-crystal-crystal protein film voltammetry and by in situ STM to single-molecnle resolntion. Top left P.

As for iyrd, the combination of voltammetry and in situ STM has thus addressed a standing issue in protein film voltammetry, viz. the fraction of immobilized protein molecules that retains full functionality on surface immobilization. Two other observations illuminate this. One is that the enzyme function is more subtle and sensitive to non-native environments than simpler electron transfer function. Retaining full CuNiR catalytic... 

MCD spectroscopy has been used to probe the excited state electronic structure of the electron transfer relevant [Mo -OH][Fe ] state of chicken SO and the Mo(v) resting state of YedY. YedY(MsrPQ) possesses the SUOX fold, and has recently been shown to be a periplasmic methionine sulfoxide reductase. Protein film voltammetry has been used to propose a mechanism for... 

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