Intramolecular protein redox

In applying this principle to proteins, one would ideally like to modify a protein at one specific site with a number of related, substitution-inert, inorganic redox reagents, and then study the intramolecular electron transfer step as a function of a wide variety of variables (e.g., the redox potential and hydrophobicity of the redox reagent). Such a study is extremely difficult to carry out with large proteins, and none has been reported thus far. We have, however, found out that horseheart cytochrome c is amenable to modification at a single site by the... 

Although electron transfers in biological systems are generally expected to be non-adiabatic, it is possible for some intramolecular transfers to be close to the adiabatic limit, particularly in proteins where several redox centers are held in a very compact arrangement. This situation is found for example in cytochromes C3 of sulfate-reducing bacteria which contain four hemes in a 13 kDa molecule [10, 11], or in Escherichia coli sulfite reductase where the distance between the siroheme iron and the closest iron of a 4Fe-4S cluster is only 4.4 A [12]. It is interesting to note that a very fast intramolecular transfer rate of about 10 s was inferred from resonance Raman experiments performed in Desulfovibrio vulgaris Miyazaki cytochrome Cj [13]. 

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. 

Finally, intramolecular electron transport occurs in multicentre redox proteins, and represents a rather different situation. 

Examples of this mechanism are the reduction of o-nitrophenol, p-nitrosophenol, uranium complexes, and tocopherols, among many others [55], and also proteins containing two redox sites including the possibility of an intramolecular electron transfer [35]. 

A large number of enzyme activities have been shown to be affected by protein S-thiolation with glutathione redox buffers in vitro (reviewed in ref. [271]). Extracellular and cell-surface proteins are commonly activated by the formation of intramolecular disulfides. Conversely, most of the responsive intracellular enzymes are down-regulated by the formation of mixed disulfides, with the interesting exception of enzymes involved in the delivery of free glucose. 

It is well known that the redox responsiveness in proteins may be conferred by S-NO (nitrosylation), S-OH (sulfenic acid), S-S (intramolecular disulfide), and S-SR (mixed disulfide or S-thiolation), all potential reversible modifications of reactive cysteines (Figure... 

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]. 

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