Redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress. Although electron paramagnetic resonance (EPR) is extremely valuable in studying hemoglobin redox reactions it has not been adequately used. We have focused in this review on the important contributions of EPR to our understanding of hemoglobin redox reactions. We have limited our discussion to the redox reactions thought to occur under physiological conditions. This includes autoxidation as well as the reactions of hydrogen peroxide generated by superoxide dismutation. We have also discussed redox reactions associated with nitric oxide produced in the circulation. We have pinpointed the value of using EPR to detect and study the paramagnetic species and free radicals formed during these reactions. We have shown how EPR not only identifies the paramagnetic species formed but can also be used to provide insights into the mechanism involved in the redox reactions.

Periyakaruppan Thangiah Manoharan

free radical

Hemoglobin

Hydrogen peroxide

Nitric oxide

superoxide dismutase

AUTOOXIDATION

chemical reaction

circulation

electron spin resonance

erythrocyte

Human

oxidation reduction reaction

oxidative stress

oxygen tension

oxygenation

priority journal

review

IIT Madras is a public technical and research university located in Chennai, Tamil Nadu. Founded in 1959, it is recognised as an Institute of National Importance.

IIT Madras has been ranked as the top engineering institute in India for four years in a row by the National Institutional Ranking Framework of the MHRD

It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Redox reactions of hemoglobin.

Antioxidants and Redox Signaling

The role of the β-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a g(iso) of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the β-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the β-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the β-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.

Biochemistry

Superoxide produced in the heme pocket of the β-chain of hemoglobin reacts with the β-93 cysteine to produce a thiyl radical

By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g is parallel with = 2.0565 and g is perpendicular to = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with ((O17-O17))A is perpendicular to = 63 G and ((O17- O16)A is perpendicular to = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket.

Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions

EPR Detection of Glutathiyl and Hemoglobin-cysteinyl Radicals during the Interaction of Peroxynitrite with Human Erythrocytes†

Biophysical Journal

Comparative Study of Tyrosine Radicals in Hemoglobin and Myoglobins Treated with Hydrogen Peroxide

Site-Specific Cross-Linking of Human and Bovine Hemoglobins Differentially Alters Oxygen Binding and Redox Side Reactions Producing Rhombic Heme and Heme Degradation†

Redox Reactions of Hemoglobin

Journal	Antioxidants and Redox Signaling
Publisher	Mary Ann Liebert Inc.
ISSN	15230864
Open Access	No