Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss

Abstract

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.

Figure 1

Mechanism of iron oxidation in heme. The heme iron can be oxidized in two mechanisms: when the concentration of O₂ is high (the top method) or low (the bottom method) (Adapted from 14). Under high concentrations of O₂ (1), a hydronium molecule bonds with O₂ and the ligand leaves as a neutral superoxide radical. A water can then hydrogen bond with the distal His. Under low concentrations of O₂ (2), a water molecule can displace the ligand. Reentry of O₂ can remove an electron from the heme iron in which the coordinated water facilitates the removal of the iron electron to O₂. The ligand leaves the heme pocket as superoxide anion radical. In both scenarios the iron heme is oxidized to Fe(III).

Figure 2

(A) Tetrameric structure of bovine Hb at pH 5.7 (heme groups are shown in red). (B) Highlighted amino acid differences in the E helix and CD turn. The structure of the trout IV β subunit is shown. The amino acid residues listed in Table VI are highlighted in red and labeled. The C, D, E, and F helices and the CD turn are labeled along with the heme and proximal and distal histidine residues.

Figure 3

E11 positioning in the bovine, trout IV, and perch Hb. The CO-bound bovine at pH 8.5, the met-trout IV at pH 6.3, and the met-perch at pH 8.0 structures are shown for the A) α and B) β subunits. The CO-bound bovine at pH 8.5 was overlaid onto the trout IV and perch Hbs to place the CO (red) and calculate ligand distances to the E11 residue. The met-structure of the bovine, trout IV, and perch at pH 5.7 are shown for the C) α and D) β subunits with the water above the heme iron represented as a blue sphere. The E11 residues and distances to the ligand are listed for A–D). The two α and β subunits of each Hb structure were overlaid onto one another. The Ile in the perch α structure differs in conformation between the two α subunits.

Figure 4

E10 Lys interactions with the distal His and heme propionate atoms in bovine Hb. A) The large positively charged portion of E10 interacts with the heme propionate oxygen atom and the distal His through separate water molecules in bovine Hb at pH 6.3 in the α subunit. B) E10 interacts directly with the distal His in the bovine Hb at pH 8.5 in the β subunit.

Figure 5

Structural differences in the heme pocket of bovine, trout IV, and perch Hb. Structural differences of heme pocket residues are shown for the bovine Hb at pH 8.5, the trout IV Hb at pH 6.3, and the perch Hb at pH 8.0 are shown for the A) α and B) β subunits. The CO-bound bovine at pH 8.5 was overlaid onto the trout IV and perch Hbs to place the CO (in the red stick model). The residues are labeled accordingly and contact distances to the heme propionates are listed in black for the structures shown and in parentheses calculated using the structures at pH 5.7 for all three Hbs.