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CHAPTER 1

OVERVIEW

 

The temperature dependence of the formal potential and the rate constant of the heterogeneous electron transfer reaction of myoglobin has been investigated using cyclic voltammetry. Evidence for the non-spontaneous disassociation of the heme from the protein followed by the spontaneous complete unfolding and denaturation of the protein is indicated by a radical change in the reaction center entropy (D Src) at 37° C. Furthermore from 5-37° C, the cyclic voltammograms display trends consistent with the gradual increase in the reversibility of the reaction. From 37-50° C, the cyclic voltammograms show a switch to more irreversible reactions. The electrochemistry of myoglobin will be analyzed and discussed in context with analogous experimental studies which have been performed on ferricyanide and cytochrome c.

Chapter 2 is a structural and functional overview of myoglobin. Found primarily in cardiac and red skeletal muscles, myoglobin functions in the storage of oxygen and facilitates the transport of oxygen to the mitochondria for oxidative phosporylation. The experiments which elucidated the three-dimensional structure of myoglobin will be discussed. As the capacity of myoglobin to bind oxygen depends on the presence of a heme, a review of the heme will be offered. Additionally, stability studies as well as a conformational analysis of myoglobin will be featured.

The focus will shift to electrochemistry. If an electrode is at equilibrium with the solution in which it is immersed, the electrode will have a potential, invariant with time, which is thermodynamically related to the composition of the solution. In solution, species O is capable of being reduced to R at the electrode by the following reversible electrochemical reaction:

O + ne- Û R

Cyclic voltammetry is one of the most versatile electroanalytical techniques for the study of electroactive species. Cyclic voltammetry involves the cycling of the potential of an electrode, which is immersed in an unstirred solution, and measuring the resulting current. The voltammogram is a display of current versus potential. A model voltammogram will be dissected ensuring a clear understanding of the studies performed here. Specifically, a potentiostat applies a potential to the electrochemical cell, usually a three-electrode configuration, and a current to voltage converter measures the resulting current. This instrumentation section will examine the reference electrode and discuss a simple procedure used to measure its potential. Other highlights include a discussion of ohmic potential drop, mass transfer, and the electrical double-layer. Finally, ferricyanide is a model system commonly used in electrochemistry which can be described by the reversible process:

[FeII(CN)6]3- + e- Û [FeIII(CN)6]4-

Results for the effect of scan rate, concentration, and supporting electrolyte on cyclic voltammograms for ferricyanide will be analyzed.

Oxidative phosphorylation takes place in the inner mitochondrial membrane. Mitochondria are the site of aerobic respiration within the cell. Mitochondrial cytochromes c are the most extensively studied electron-transfer proteins. A structural and functional overview of cytochrome c is the main topic of Chapter 4. Briefly, the various confromations of cytochrome c which have been studied will be introduced.

Chapter 5 links the biochemistry with the electrochemistry. Topics include semiconductors, biological membranes, stability, and reduction potentials. The study of heterogeneous electron transfer reactions involves the study of electron transfer between a solid protein and a substance in solution. A typical cyclic voltammogram and an illustration of the electrode-potential complex of cytochrome c will be shown. The electrochemical behavior of myoglobin has been characterized as a quasi-reversible response with reaction rates of oxidation being much slower than the reduction. Myoglobin/oxygen ligand-binding reactions studied directly at indium oxide transparent electrodes suggest the reaction mechanism is an EC mechanism (an electrode reaction followed by a chemical reaction that consumes the product of the electrode reactions ) as shown below:

Electrode: Mb(III) + e- Û Mb(II)

Solution: Mb(II) + O2 Û Mb(II)O2

Moreover, the site of electron transfer is buried with respect to the protein surface. Typical voltammograms of myoglobin will be displayed.

The design of the electrochemical cell especially designed for temperature studies will be detailed in Chapter 6. Following will be a presentation of temperature studies conducted for ferriyanide and cytochrome c. Finally, the temperature dependence of the formal potential and the rate constant of the heterogeneous electron transfer reaction of myoglobin will be analyzed.