The peroxidatic activities of Myoglobin and Hemoglobin, their pathological consequences and possible medical interventions

Abstract

Under those pathological conditions in which Myoglobin and Hemoglobin escape their cellular environments and are thus separated from cellular reductive/protective systems, the inherent peroxidase activities of these proteins can be expressed. This activity leads to the formation of the highly oxidizing oxo-ferryl species. Evidence that this happens in vivo is provided by the formation of a covalent bond between the heme group and the protein and this acts as an unambiguous biomarker for the presence of the oxo ferryl form. The peroxidatic activity also leads to the oxidation of lipids, the products of which can be powerful vasoconstrictive agents (e.g. isoprostanes, neuroprostanes). Here we review the evidence that lipid oxidation occurs following rhabdomyolysis and sub-arachnoid hemorrhage and that the products formed from arachidonic acid chains of phospholipids lead, through vasoconstriction, to kidney failure and brain vasospasm. Intervention in these pathological conditions through administration of reducing agents to remove ferryl heme is discussed. Through-protein electron transfer pathways that facilitate ferryl reduction at low reductant concentration have been identified. We conclude with consideration of the therapeutic use of Hemoglobin Based Oxygen carriers and how the toxicity of these may be reduced by engineering such electron transfer pathways into hemoglobin.

Keywords: Blood substitute; Ferryl; Hemoglobin; Myoglobin; Peroxidase; Rhabdomyolysis.

Fig. 1

Reverse-phase HPLC of myoglobin (red line) and oxidatively modified myoglobin (blue line). Myoglobin shows heme B (∼14.7 min) and apoMb (22.1 min) only. Peroxide-damaged Mb shows additional oxidatively modified hemes (7–14 min) and a heme that is covalently attached to the globin (peak maximum 23.7 min). Inset: HPLC optical spectra of unmodified heme (A, red) oxidatively modified ‘d-type’ free heme (B, blue) and covalently bound cross-linked heme (C, green). Spectra are offset for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Scheme showing the basic mechanism of ferric-ferryl redox cycling in lipid-based oxidative damage. Ferryl heme iron (and also protein-based radicals) can oxidize lipid membranes to initiate formation of a series of isoprostanes (15-F₂ Iso-P shown) with similar vasoactive properties to prostaglandins. Often resulting in acidosis due to the reduced supply of oxygen to the tissue, the lowered pH enhances and amplifies lipid oxidation in a vicious cycle.

Fig. 3

(A) CP20 acts as a reductant for ferryl myoglobin (sperm whale), similar to urate and ascorbate, reducing the protein to the ferric oxidation state. (B) The kinetic profile of the reduction of ferryl myoglobin by CP20 (circles). The profile is fitted to a two hyperbola representing the two possible electron pathways. The through-protein pathway can be eliminated through mutation of the myoglobin to remove a redox active tyrosine between the heme iron and external environment (Tyr103 to Phe, squares).

Fig. 4

Two site model for reduction of ferryl globin. Through-protein electron transfer via redox active tyrosine (top left panel) results in a high affinity pathway enhancing ferryl reduction at low concentrations of reductant (lower panel circled in green). Direct access to the heme pocket (top right panel) results in a lower affinity electron pathway (lower panel circled in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Aplysia myoglobin exhibits extensive enhancement of the ferryl reduction kinetics by reductants upon selected tyrosine mutations. Left: Crystal structure of myoglobin from Aplysia faciata (Aplysia limacina as a junior synonym). PDB code 1MBA, this wild type protein was mutated (V63H, F42Y and F98Y) in silico. This protein has 15 phenylalanine residues close to the heme iron that can be selectively mutated to redox active tyrosine residues. Right: The V63H variant (green) was used to provide a distal histidine present in most human globins. V63H/F42Y (purple) and V63H/F98Y (blue) mutations exhibits significantly enhanced kinetics of ferryl reduction by low micromolar levels of reductants (50 μM Ascorbate shown). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Sites of mutations introduced on β subunit of hemoglobin to enhance ferryl reduction by reducing agents. Each site on the beta chain (blue ribbon) adds a redox active tyrosine to shuttle electrons from a reductant to the heme iron (β-F41Y, β-K66Y, β-F71Y, β-T85Y, β-F85Y, β-F91Y, β-F96Y). The alpha chain (green ribbon) already has this pathway (α-Y42) Hemes shown in stick form.PDB 1HHO was used to construct this image with the two images rotated by 90°. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)