Molecular controls of the oxygenation and redox reactions of hemoglobin

Abstract

Significance: The broad classes of O(2)-binding proteins known as hemoglobins (Hbs) carry out oxygenation and redox functions that allow organisms with significantly different physiological demands to exist in a wide range of environments. This is aided by allosteric controls that modulate the protein's redox reactions as well as its O(2)-binding functions.

Recent advances: The controls of Hb's redox reactions can differ appreciably from the molecular controls for Hb oxygenation and come into play in elegant mechanisms for dealing with nitrosative stress, in the malarial resistance conferred by sickle cell Hb, and in the as-yet unsuccessful designs for safe and effective blood substitutes.

Critical issues: An important basic principle in consideration of Hb's redox reactions is the distinction between kinetic and thermodynamic reaction control. Clarification of these modes of control is critical to gaining an increased understanding of Hb-mediated oxidative processes and oxidative toxicity in vivo.

Future directions: This review addresses emerging concepts and some unresolved questions regarding the interplay between the oxygenation and oxidation reactions of structurally diverse Hbs, both within red blood cells and under acellular conditions. Developing methods that control Hb-mediated oxidative toxicity will be critical to the future development of Hb-based blood substitutes.

FIG. 1.

A cartoon representation of the evolutionary appearance of different heme proteins associated with the hemoglobins (Hbs). The vertical scale gives the time in millions of years when the respective proteins evolved. The top panel shows the gene that encodes different heme proteins. The E_1/2 values are versus NHE. The data for myoglobin (horse heart Mb) and Hb (Hb A) are from Ref. (114). Conditions: 0.05 M 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.1. E_1/2 values of individual α and β-Hb chains are from Ref. (27). Conditions: 1 M Glycine buffer at pH 6 at 6°C.

FIG. 2.

Cartoon showing a similar in-plane movement of heme iron on oxygenation (left) and oxidation (right). (A) A schematic representation of the movement of the Fe²⁺ center in Hb associated with the oxygenation process. The deoxy (T-state) Hb having low O₂ affinity has a high spin Fe²⁺ electron configuration with five ligands. On O₂ binding, a change in spin state to low spin Fe²⁺ leads to a more planar oxy (R-state) Hb with higher O₂ affinity. The vertical scale on the left shows the change in ionic radius of Fe²⁺ on changing from the high spin to low spin state. (B) A similar movement of iron on oxidation from Fe²⁺ deoxy T-state to aquomet R-like Fe³⁺ state and the vertical scale on the left represents the E_1/2 values (vs. NHE) for T- and R-state Hb, respectively. The E_1/2 values quoted in (B) are for Hb A in the presence of a large excess of inositol hexaphosphate (IHP) and NaNO₃ (T-state stabilized) and carboxypeptidase digested Hb A (R-state stabilized) (42). Conditions: 0.05 M MOPS at pH 7.1.

FIG. 3.

Hill plots for Hb A and hMb under varied conditions. Plot reproduced from Ref. (41) and used with permission. Symbols: open circles, hMb in 0.05 M MOPS, 0.2 M NaNO₃ (log P₅₀=−0.48); closed circle, Hb A in 0.05 M MOPS (log P₅₀=0.15); open triangles, Hb A in 0.05 M MOPS, 0.2 M NaNO₃ (log P₅₀=−0.87); closed triangles, Hb A in 0.05 M MPOS, 0.2 M NaNO₃, 16-fold excess IHP (log P₅₀=+1.55). The log P₅₀ values show a gradual increase toward higher partial pressure of oxygen from hMb to Hb A in the presence of the T-state stabilizer IHP, representing greater difficulty of oxygenation for T-state stabilized heme proteins. Experiments were performed at neutral pH and at room temperature.

FIG. 4.

Nernst plot of Hb A and hMb under different conditions obtained from spectroelectrochemistry experiments at 20°C at neutral pH. The potentials are reported versus Ag/AgCl electrode. Plot reproduced from Ref. (41) and used with permission. Symbols: open circles, hMb in 0.05 M MOPS, 0.2 M NaNO₃ (E_1/2=−160 mV); closed circle, Hb A in 0.05 M MOPS (E_1/2=−104 mV); open triangles, Hb A in 0.05 M MOPS, 0.2 M NaNO₃ (E_1/2=−63 mV); closed triangles, Hb A in 0.05 M MOPS, 0.2 M NaNO₃, 10-fold excess IHP (E_1/2=−40 mV). The E_1/2 values (vs. Ag/AgCl) show a gradual shift toward more positive values as the heme protein conformation changes from R-to-T state, indicating greater ease of reduction for T-state stabilized heme proteins. See text for more explanation.

FIG. 5.

A qualitative energy-level diagram showing the relative energies of Fe²⁺-Hb and Fe³⁺-Hb from two different hypothetical Hb samples (represented by solid and dashed lines). The points C and D represent the activated complexes for the two Hb samples that are formed in the course of auto-oxidation as discussed in the text. E^AC_act. and E^AD_act. are measures of how fast the auto-oxidation process would be in these two hypothetical Hb samples. For clarity, the energies of the Fe²⁺-Hb and Fe³⁺-Hb from two different species are considered unaltered between species. See text for more explanation.

FIG. 6.

A schematic representation of pathways for lipid oxidation and pathological complications resulting from oxidation of heme proteins (Hb and Mb), adapted from Ref. (95). The various pathways shown are as follows: Auto-oxidation of oxy-ferrous Hb can produce ferric-Hb (a). This ferric Hb can enter a redox cycle in the presence of peroxide, producing ferryl Hb and protein radicals (b). These protein radicals can combine to produce cross-linked heme-protein products (c), which accelerates lipid oxidation (d). Both native and “modified” ferryl heme proteins can initiate lipid oxidation at the membrane by the abstraction of a proton that subsequently can lead to vasoconstriction, acidosis, and pathological complications (e).

FIG. 7.

Schematic representation of parallel pathways of autoxidation in heme proteins. The heme proteins are represented as Fe²⁺ or Fe³⁺ for clarity. In pathway 1, with a high concentration of O₂, the bound O₂ is protonated and leads to oxidation of the Fe²⁺ center to Fe³⁺, followed by dissociation of peroxide radical. Water coordinates to the Fe³⁺ center, which is H-bonded with distal histidine (His). In pathway 2, under low O₂ partial pressure, water can replace the bound O₂/ligand. Re-entry of O₂ leads to oxidation of Fe²⁺ to Fe³⁺ in the heme protein, which is facilitated by the coordinated water, and O₂ leaves as a superoxide radical anion. Figure reproduced from Ref. (10) and used with permission.