Structural and Functional Studies Indicating Altered Redox Properties of Hemoglobin E

Abstract

Hemoglobin (Hb) E (␤-Glu26Lys) remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms; for example, EE individuals exhibit a mild chronic anemia, and HbE/␤-thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac dysfunction.The purpose of this study was to determine and evaluate structural and functional consequences of the HbE mutation that might account for the pathophysiology. Functional studies indicate minimal allosteric consequence to both oxygen and carbon monoxide binding properties of the ferrous derivatives of HbE. In contrast, redoxsensitive reactions are clearly impacted as seen in the following: 1) the ϳ2.5 times decrease in the rate at which HbE catalyzes nitrite reduction to nitric oxide (NO) relative to HbA, and 2) the accelerated rate of reduction of aquometHbE by L-cysteine (L-Cys). Sol-gel encapsulation studies imply a shift toward a higher redox potential for both the T and R HbE structures that can explain the origin of the reduced nitrite reductase activity of deoxyHbE and the accelerated rate of reduction of aquometHbE by cysteine. Deoxy-and CO HbE crystal structures (derived from crystals grown at or near physiological pH) show loss of hydrogen bonds in the microenvironment of ␤Lys-26 and no significant tertiary conformational perturbations at the allosteric transition sites in the R and T states. Together, these data suggest a model in which the HbE mutation, as a consequence of a relative change in redox properties, decreases the overall rate of Hb-mediated production of bioactive NO.

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

“…A recent in vitro study demonstrates for the first time that free a-globin chains are oxidatively unstable and damaging to HbA and HbE, with ferryl(Fe 4+ )HbE significantly less able to autoreduce than ferryl HbA (178). This should also be considered in light of the fact that HbE compared with HbA exhibits differences in its redox properties with indications of an increase in its redox potential (152).…”

“…A plausible mechanism for the functional impact of this local mutation at the homodimer interface is through an alteration of the flexing dynamics of homodimers, which could alter the accessibility and stability of bound and free ligands, including water within the distal heme pocket, through the modified dynamics of the nearby amino acid side chains. Overall, the greater mechanical, oxidative (106), and thermal instability (33,142) of HbE as reported in vitro may be attributed, at least in part, to the conformational changes observed at bHis116 and bHis116, communicated by the bLys26 substitution with an ensuing electrostatic change (152).…”

“…Significantly noted for COHbE is the loss of stabilizing H-bonds from bLys26 (E26) to bArg30 (R30), residues critical for Hb assembly, stability, and oxidation state. Details are presented in Roche et al (152). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars (Fig.…”

“…The hemes for the R state structure have, relative to the T state structure, a lower redox potential, which favors formation of the ferric heme product. Interestingly and highly relevant to the mechanistic enigma of HbE/b-thal are (i) HbE has a higher redox potential compared with HbA and (ii) HbE in vitro is less efficient, relative to HbA, at generating NO through the NR reaction (149,152) (Fig. 8).…”

“…This article presents an overview, without exhausting the literature, of HbE/b-thal clinical manifestations; current treatments; evidence of pathophysiological mechanisms involving oxidative stress; and new findings pointing to possible pathophysiological mechanisms involving the interplay between unstable HbE exacerbated by free a-globin chains, limited NO production as a result of HbE reduced in function as a nitrite reductase (NR) (152), enhanced population of ferryl Hb RBC damage, and lysis resulting in chronic inflammation and cardiovascular pathophysiology. Based on these insights, we FIG.…”