14] Electron paramagnetic resonance spectroelectrochemical titration

Abstract

The use of spectro-electrochemical methods to elucidate the energetics of biological electron transfer reactions has become common practice among researchers interested in characterizing the redox properties of biological systems. Simply stated, spectro-electrochemistry is the combined use of redox potentiometry and spectroscopy to determine the important thermodynamic parameters of an electron transfer reaction. These parameters include the formal potential value for the redox reaction (E°'), the number of electrons transferred in the reaction (n), and the free energy change for the reaction (AG°'). Aside from the obvious importance in understanding the energetics of single or multiple electron transfer reactions in a biological system, there are other interesting aspects to this kind of investigation. For example, the technique can be used to determine the effect that substrate or product binding has on the formal potential values of enzyme systems and to determine the binding constants for these interactions. The technique can also be used to make comparisons between different proteins containing a similar redox active center, to determine the magnitude to which different structural components around the redox active site affect the energetics of electron transfer for that particular type of center. Similarly, comparisons between the redox potential values of proteins subjected to site-directed mutations around the redox active site enable one to ascertain the importance of various amino acid residues for the energetics of that electron transfer reaction. Some innovative spectro-electrochemical methods have included the use of visible and electron paramagnetic resonance (EPR) spectroscopies for the quantitation of redox species present during potentiometric titration.