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. 2011 Jul 1;286(26):23452-66.
doi: 10.1074/jbc.M110.183186. Epub 2011 Apr 29.

Structural and functional studies indicating altered redox properties of hemoglobin E: implications for production of bioactive nitric oxide

Camille J Roche  1 , Vladimir Malashkevich, Tatiana C Balazs, David Dantsker, Qiuying Chen, Juan Moreira, Steven C Almo, Joel M Friedman, Rhoda Elison Hirsch
Affiliations

Structural and functional studies indicating altered redox properties of hemoglobin E: implications for production of bioactive nitric oxide

Camille J Roche et al. J Biol Chem. .

Abstract

Hemoglobin (Hb) E (β-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/β-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, redox-sensitive reactions are clearly impacted as seen in the following: 1) the ∼2.5 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 βLys-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.

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Figures

FIGURE 1.
FIGURE 1.
Structural superposition of the human (h) HbE structures in the vicinity of βLys-26. Top panel, structural environment of βLys-26 (K26) in the high salt HbE superimposed onto the experimental omit electron density map. α chain is shown in light green, and β chain is shown in gray. Conformations of βLys-26 and nearby residues in high salt (gray), low salt (two subunits, salmon), and deoxy (two subunits, blue) HbE structures are shown. Bottom panel, structural superposition between the COHbA and COHbE structures in the area of β-Glu26Lys mutation. α chain is shown in light green, and β chain is shown in gray. Water molecules in the COHbA structure are shown as red spheres. Side chains corresponding to the COHbA structure are depicted in pink. Note hydrogen bond between βGlu-26 (E26) and βArg-30 (R30).
FIGURE 2.
FIGURE 2.
Time-dependent change in the CO recombination trace for samples of COHbE and COHbA encapsulated in a thin sol-gel matrix. The samples were initially encapsulated as the deoxy derivatives and subsequently converted to the CO derivative after allowing the sol-gel to age for several days in an oxygen-free environment. The bathing buffer of the sol-gel films is 0.05 m BisTris OAc, pH 7.0. The progression of traces tracks the normalized absorbance (N.A.) changes in the recombination profile, starting from the initial exposure of the deoxyHb samples to CO binding, laser-induced CO photodissociation, and recombination (see “Experimental Procedures”).
FIGURE 3.
FIGURE 3.
Comparison of the initial rate for the nitrite reductase reaction for HbA and HbE in solution at pH 7.0 (0.2 mm heme + ∼1:1 nitrite, 0.05 m BisTris OAc, pH 7.0, with 1 mm dithionite) as reflected in the decrease in the deoxyHb OD at 430 nm. The right panel shows the change in the Sôret absorption band as a function of time for HbA over the same time period. Solution conditions are as described in the text. The down-pointing arrow indicates the time-dependent decrease in absorbance at 430 nm.
FIGURE 4.
FIGURE 4.
Time-dependent changes in the visible absorption spectrum reflecting the nitrite reductase reaction for sol-gel-encapsulated Hbs trapped in the T state (0. 4 mm heme + 5 mm nitrite, 0.05 m Bistris OAc, pH 7.4, + 1 mm dithionite). The upward and downward pointing arrows indicate the increasing and decreasing absorbance, respectively. Inset, representative plot of the time-dependent changes in the population of different HbA species resulting from the nitrite reductase reaction of the sol-gel sample. (See “Experimental Procedures” for details). Red circle, NOHb; black square, deoxy; blue triangle, metHb.
FIGURE 5.
FIGURE 5.
Comparison of the initial rate for the nitrite reductase reaction for HbA in solution in the presence and absence of L35. DeoxyHbA + 1:1 nitrite ± L35. Red trace, 0.22 mm heme + L35 (1:1 heme); blue trace, 0.22 mm heme without L35. Solution conditions are as follows: 0.05 m BisTris, pH 7.4; arrows represent approximate time points when Band III peaks were generated (see Fig. 6).
FIGURE 6.
FIGURE 6.
Changes in the normalized absorption spectrum of Band III in the reaction of deoxyHbA with nitrite and at the appearance of the change in slope for this reaction. Black line, deoxy T state at time 0; red line, Band III at time point at the change in slope. Inset shows the time progression of Band III.
FIGURE 7.
FIGURE 7.
Absorption changes of the R state deoxyHbA sol-gel + 0.5 mm nitrite as a function of time; 0.05 m BisTris, pH 7.4, 0.45 mm (heme). Inset A is the distribution of deoxy as a function of time; inset B is the initial position of Band III.
FIGURE 8.
FIGURE 8.
Kinetic trace of the reduction of metHb by l-cysteine in solution: (metHb (0. 4 mm heme) + 2 mml-Cys, PBS, pH 7.4). l-Cys is added to samples of metHb anaerobically to initiate reaction. The samples are then scanned at 2-min intervals, and base-line corrected. The change in 630 nm (unique metHb band) is monitored as a function of time. Inset, a representative plot showing the increase and decrease in the spectral bands of HbA as a function of time are indicated by up- and down-pointing arrows, respectively.
FIGURE 9.
FIGURE 9.
Kinetic trace of the reduction of metHb by l-Cys in sol-gels. 0.4 mm (heme) + 10 mm l-Cys, PBS, pH 7.4. Samples of Hb encapsulated as T (deoxy) or R (ligated to CO) state are converted to met as described under “Experimental Procedures.” l-Cys is added to initiate reaction and monitored as in solution (Fig. 5). The shorter x axis scale (seconds) for the T state samples was compared with the x axis used on the R state samples serves to highlight the difference in the rate between the T and R states and the biphasic nature of the data. a, HbA; b, HbE.
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