EPR in chemistry

Enzyme Reactions

Detection and study of the active site of Cu,Zn-SOD.
Many enzyme reactions involve one-electron oxidation steps with formation of paramagnetic transient state of the enzyme detectable by EPR. The paramagnetic center where the unpaired electron is located, is usually centered at a transition metal (metalloproteins) or is an amino acid derived radical. Detection and identification of the paramagnetic centers is important to understand the function of the enzymes. For example, in the native SOD1 enzyme, the active site contains one Cu(II) ion that gives a very characteristic EPR spectrum.

Crystal structure (1E9P.pdb) of Cu(II)-SOD protein

EPR spectrum of Cu(II)-SOD protein at 77 K using finger dewar

Reaction Kinetics

Kinetics analysis of vitamin C antioxidant ability.
Many chemical reactions involve the transfer of one electron. Each electron transfer results in an unpaired electron creating paramagnetic free radicals. EPR is the ideal spectroscopic technique to measure these species as well as to monitor the time behavior of their creation and disappearance. EPR solely has the ability to detect free radicals unambiguously. For example, antioxidants such as vitamin C are important in neutralizing dangerous free radicals in living things and kinetics indicates their effectiveness.

Experimental data of the reduction of the nitroxide TEMPOL by ascorbate (vitamin C)

Mechanism of TEMPOL reduction by vitamin C

Photochemistry

Light degradation of hops in beer.
The majority of photochemical reactions take place through free radical formation as intermediates. For example, hops used in the brewing process contain a mixture of active components that include humulones, cohumulones, adhumulones, beta acids and essential oils. Some forms of these components are photo-active. Light exposure of beer leads to the formation of free radicals that combines with sulfur compounds and gives unpleasant flavor and odor to the beer.

_{The hop product was exposed to UV/Vis light from 220-600 nm using UV lamp accessory. The light-induced free radicals were recorded in the presence of the spin trap DMPO and identified as superoxide anion radical and two C-centered radicals.}

Catalysis

Hydroxyl radical generation through photocatalytic reaction of TiO₂.
The modern chemical industry relies heavily on homogeneous and heterogeneous catalysts. Understanding the operational mode, or reactivity, of these catalysts is crucial for improved developments and enhanced performance. Where paramagnetic centers are involved, ranging from transition metal ions to defects and radicals, EPR spectroscopy is without doubt the technique of choice. For example, photocatalytic oxidation of organic pollutants is frequently carried out using semiconducting polycrystalline powders such as TiO₂. A hydroxyl radical is easily formed by light irradiation of TiO₂ and detected by EPR using spin traps.

Mechanism of hydroxyl radical formation upon light irradiation of TiO2

EPR spectra obtained upon irradiation of aqueous TiO2 suspensions in the presence of spin trapping agent PBN

Electrochemistry

EPR electrochemistry study of ruthenium complexes.
Electrochemical generation method combined with EPR has been used to identify and investigate free radicals derived from both organic and inorganic compounds. Inorganic dyes can be used to improve the efficiency of solar cells. In order to optimize the ligands, one must understand the electronic structure of the dye. Here the electrochemistry and EPR combined with DFT calculations and UV/Vis spectroscopy show the unpaired electron is delocalized between the metal and the ligand.

Data courtesy of Prof. J. Rochford, University of Massachusetts Boston (Inorg. Chem., 2016, 55 (5), pp 2460–2472)

Redox Chemistry

Enzymatic activity of SOD protein studied via Cu(II) reduction.

Enzymes in the human body regulate oxidation-reduction reactions. These complex proteins, of which several hundred are known, act as catalysts, speeding up chemical processes in the body. Oxidation-reduction reactions also take place in the metabolism of food for energy, with substances in the food broken down into components the body can use. For example, the dismutase activity of Cu,Zn-SOD protein involves reduction of Cu(II)-SOD to Cu(I)-SOD:

Reduction of Cu(II)-SOD (EPR active) to Cu(I)-SOD (EPR inactive)

Cu(II)-SOD has a very characteristic EPR signal which decays upon reduction of Cu(II) -> Cu(I).

Antioxidants

Detection of ascorbate radical upon oxidation of vitamin C.
The delicate balance between the advantageous and detrimental effects of free radicals is one of the important aspects of human (patho)physiology. Imbalanced generation of toxic radicals is highly correlated with the pathogenesis of many diseases which require the application of selected antioxidants to regain the homeostasis. EPR is used to determine the oxidative status of biological systems using endogenous long-lived free radicals (ascorbyl radical, tocopheroxyl radical, melanin) as markers.

Reaction of a toxic radical R● with an antioxidant A. Also pictured is the reaction of the antioxidant ascorbic acid (Vitamin C) with a radical

EPR spectrum of ascorbate (Vitamin C) radical