Proteins redox reactions, intramolecular

In biological systems, electron transfer kinetics are determined by many factors of different physical origin. This is especially true in the case of a bimolecular reaction, since the rate expression then involves the formation constant Kf of the transient bimolecular complex as well as the rate of the intracomplex transfer [4]. The elucidation of the factors that influence the value of Kf in redox reactions between two proteins, or between a protein and organic or inorganic complexes, has been the subject of many experimental studies, and some of them are presented in this volume. The complexation step is essential in ensuring specific recognition between physiological partners. However, it is not considered in the present chapter, which deals with the intramolecular or intracomplex steps which are the direct concern of electron transfer theories. 

Electroanalyhcal techniques (also in combination with other techniques, e.g., ophcal techniques such as photometry and Raman spectrometry) can be employed to inveshgate many functional aspects of proteins and enzymes in particular. It is possible to study the biocatalytic process with respect to the chemistry of the active site, the interfacial and intramolecular ET, slow enzyme achva-tors or inhibitors, the pH dependence, the transport of tlie substrate, and even more parameters. For example, slow scan voltammetry can be used to determine the relation of ET rates or of protonation and ligand binding. In contrast, fast scan voltammetry allows the determination of rates of interfacial ET. In addition, it is also possible to investigate chemical reactions that are coupled to the ET process, such as protonation. The use of direct ET for mechanistic studies of redox enzymes was recently reviewed by Leger and Bertrand [27]. Mathemahcal models help to elucidate the impact of different variables on the enhre current signal [27, 75, 76]. 

The reactive site of the cysteinyl residue is the thiol group, which is deprotonated at alkaline pH (pXa around 8.5). The residue under oxidizing conditions (and neutral to alkaline pH) is able to react with a similar residue under formation of a disulfide bond. Many proteins are stabilized by intramolecular disulfide bonds (e.g., insulin, growth hormone, lGF-1), but intermolecular bonds may also result from the reaction under formation of aggregates. In order to avoid unintended disulfide bond formation/cleavage, the redox potential of the solution must be monitored and controlled. In practice, aqueous buffers contain micromolar amounts of dissolved oxygen assuring a redox potential of 200-600 mV, which is sufficient to maintain the intramolecular disulfide bonds. Proteins with free cysteines may... 

The metals are generally found either bound directly to proteins or in cofactors such as porphyrins or cobalamins, or in clusters that are in turn bound by the protein the ligands tire usually O, N, S, or C. Proteins with which transition metals and zinc are most commonly associated catalyze the intramolecular or intermolecular rearrangement of electrons. Although the redox properties of the metals are important in many of the reactions, in others the metal appears to contribute to the structure of the active state, e.g., zinc in the Cu-Zn dismutases and some of the iron in the photosynthetic reaction center. Sometimes equivalent reactions are catalyzed by proteins with different metal centers the metal binding sites and proteins have evolved separately for each type of metal center. 

These intramolecular electron transfer processes provide an opportunity to examine electron transfer within the protein environment. Addition of a reduc-tant, such as aniline, results in efficient reaction of the Ru(III) with the reduc-tant to form Ru(II), which leaves the heme iron in the reduced state. If a redox active metalloprotein is present in the solution, electron transfer between the reduced heme and the added protein can be observed. Production of reduced heme iron and removal of the Ru(III) intermediate can be accomplished within a few hundred nanoseconds, which allows the study of extremely rapid interprotein electron transfer reactions. 

Covalent attachment of ruthenium ions to proteins (ruthenation) provides a powerful approach for introducing a second redox active group into an electron transfer protein (77, 78). Under suitable conditions, intramolecular electron transfer may be monitored between Ru and the intrinsic redox group. Ruthenated proteins display a number of advantages for the study of intramolecular electron transfer reactions (77, 78) ... 

Early reports on interactions between redox enzymes and ruthenium or osmium compounds prior to the biosensor burst are hidden in a bulk of chemical and biochemical literature. This does not apply to the ruthenium biochemistry of cytochromes where complexes [Ru(NH3)5L] " , [Ru(bpy)2L2], and structurally related ruthenium compounds, which have been widely used in studies of intramolecular (long-range) electron transfer in proteins (124,156-158) and biomimetic models for the photosynthetic reaction centers (159). Applications of these compounds in biosensors are rather limited. The complex [Ru(NHg)6] has the correct redox potential but its reactivity toward oxidoreductases is low reflecting a low self-exchange rate constant (see Tables I and VII). The redox potentials of complexes [Ru(bpy)3] " and [Ru(phen)3] are way too much anodic (1.25 V vs. NHE) ruling out applications in MET. The complex [Ru(bpy)3] is such a powerful oxidant that it oxidizes HRP into Compounds II and I (160). The electron-transfer from the resting state of HRP at pH <10 when the hemin iron(III) is five-coordinate generates a 7i-cation radical intermediate with the rate constant 2.5 x 10 s" (pH 10.3)... 

Additional redox signaling reactions include intramolecular modifications of proteins by oxidation of cysteine (and methionine) residues and intramolecular disulfide bonding in monomeric proteins. Some of these reactions are readily reversible and therefore they could potentially be the most relevant for hypoxic transduction in the carotid body chemoreceptor cells. The reversibdity of these reactions makes them most suitable to switch on and off the responses to hypoxia in the CB, which are fast in onset and termination (23,24). Therefore, we will describe in some detail the reactions as well as the potential mechanisms to circumscribe them to the molecular effectors of the hypoxic response. 

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