Somasundaram Ramasamy

Redox Reactions of Hemoglobin

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.

Hemoglobin redox reactions and oxidative stress

Abstract The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.

The quaternary hemoglobin conformation regulates the formation of the nitrite-induced bioactive intermediate and the dissociation of nitric oxide from this intermediate

Deoxyhemoglobin reduces nitrite to nitric oxide (NO). In order to study the effect of the hemoglobin quaternary conformation on the nitrite reaction, we compared T-state deoxyhemoglobin with R-state deoxyhemoglobin produced by reacting hemoglobin with carboxypeptidase-A prior to deoxygenation. The nitrite reaction with deoxyhemoglobin was followed by chemiluminescence, electron paramagnetic resonance and visible spectroscopy. The initial steps in this reaction involve the binding of nitrite to deoxyhemoglobin followed by the formation of an electron delocalized metastable intermediate that retains potential NO bioactivity. This reaction is shown by visible spectroscopy to occur 5.6 times faster in the R-state than in the T-state. However, the dissociation of NO from the delocalized intermediate is shown to be facilitated by the T-quaternary conformation with a 9.6 fold increase in the rate constant. The preferred NO-release in the T-state, which has a higher affinity for the membrane, can result in the NO diffusing out of the RBC and being released to the vasculature at low partial pressures of oxygen.

pH influenced metal ion coordination changes in reconstituted hemoglobin

This paper covers a detailed analysis of the coordination changes taking place at the active sites in both Cu and Ni reconstituted hemoglobin as a function of pH. These experiments provide insight into how proteins are held in their native configuration. The EPR results of CuHb reveal that the species formed in extreme acidic condition were different from those formed at extreme basic condition. At pH 3 we see an isotropic spectrum characteristic of 4-coordinated species, while at pH 12 there is an indication of equilibrium between mixtures of species. Further support for the above coordination changes is obtained from FT-Raman of NiHb at different pH conditions. At pH 3 all the 5-coordination marker bands are lost and there is a shift in the 4-coordination marker band, while at pH 12 both 4- and 5-coordination marker bands are still seen with slight shift in their positions. In addition to this, we could see a new peak at 1633 cm−1. The coordination changes as a function of pH could be seen for both CuHb and NiHb using UV-visible spectroscopic techniques.

A new paramagnetic intermediate formed during the reaction of nitrite with deoxyhemoglobin

The reduction of nitrite by deoxygenated hemoglobin chains has been implicated in red cell-induced vasodilation, although the mechanism for this process has not been established. We have previously demonstrated that the reaction of nitrite with deoxyhemoglobin produces a hybrid intermediate with properties of Hb(II)NO+ and Hb(III)NO that builds up during the reaction retaining potential NO bioactivity. To explain the unexpected stability of this intermediate, which prevents NO release from the Hb(III)NO component, we had implicated the transfer of an electron from the β-93 thiol to NO+ producing ·SHb(II)NO. To determine if this species is formed and to characterize its properties, we have investigated the electron paramagnetic resonance (EPR) changes taking place during the nitrite reaction. The EPR effects of blocking the thiol group with N-ethyl-maleimide and using carboxypeptidase-A to stabilize the R-quaternary conformation have demonstrated that ·SHb(II)NO is formed and that it has the EPR spectrum e...