A Biophysical Investigation of Recombinant Hemoglobins with Aromatic B10 Mutations in the Distal Heme Pockets^,

Abstract

This study examines the structural and functional effects of amino acid substitutions in the distal side of both the alpha- and beta-chain heme pockets of human normal adult hemoglobin (Hb A). Using our Escherichia coli expression system, we have constructed four recombinant hemoglobins: rHb(alphaL29F), rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W). The alpha29 and beta28 residues are located in the B10 helix of the alpha- and beta-chains of Hb A, respectively. The B10 helix is significant because of its proximity to the ligand-binding site. Previous work showed the ability of the L29F mutation to inhibit oxidation. rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) exhibit very low oxygen affinity and reduced cooperativity compared to those of Hb A, while the previously studied rHb(alphaL29F) exhibits high oxygen affinity. Proton nuclear magnetic resonance spectroscopy indicates that these mutations in the B10 helix do not significantly perturb the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces, while as expected, the tertiary structures near the heme pockets are affected. Experiments in which visible spectrophotometry was utilized reveal that rHb(alphaL29F) has equivalent or slower rates of autoxidation and azide-induced oxidation than does Hb A, while rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) have increased rates. Bimolecular rate constants for NO-induced oxidation have been determined using a stopped-flow apparatus. These findings indicate that amino acid residues in the B10 helix of the alpha- and beta-chains can play different roles in regulating the functional properties and stability of the hemoglobin molecule. These results may provide new insights for designing a new generation of hemoglobin-based oxygen carriers.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

“…Interestingly, by placing an aromatic residue at the E11 position of the ␤-subunit, we generated a high O 2 affinity mutant in rHb (␤V67F). This mutant has higher oxygen affinity than that reported for rHb (␣L29F) (13,15).…”

“…2). The other three mutants bind O 2 with low affinity and have higher autoxidation rates than that of the wild type protein (15). In general, the two ␣-subunit mutants have lower azide-and NOinduced oxidation rates, whereas the two ␤-subunit mutants have a lower NO-induced but higher azide-induced oxidation rates than Hb A (6,15).…”

“…1). Hemoglobin mutants carrying an aromatic replacement at the B10 position have been characterized by several groups (6,13,15,31,52,55). Wiltrout et al (15) substituted B10Leu with either phenylalanine or tryptophan.…”

“…1). The importance of these residues on ligand binding has been studied by several groups (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

“…We and others have previously generated B10Leu mutants and shown that replacement of the ␣B10Leu residue (␣Leu-29) with phenylalanine increases the oxygen affinity and decreases the autoxidation rate of the macromolecule (6,13,15,31,32). We have also produced an octameric hemoglobin with wild type biological activities by substituting the ␣-subunit Asn-78 residue with cysteine (33).…”