A Biophysical Investigation of Recombinant Hemoglobins with Mutations in the Distal Heme Pocket

Mary Ellen Wiltrout
Carnegie Mellon University

We have investigated the effect of amino acid substitutions in the distal side of the heme pocket of both the ?- and ?-chains of human normal adult hemoglobin (Hb A) on the structural and functional properties of hemoglobin. Using our E. coli expression system, we have constructed and expressed four recombinant hemoglobins, rHb (?L29F), rHb (?L29W), rHb (?L28F), and rHb (?L28W). The ?29 and ?28 residues are located in the B10 helix of the ?- and ?-chains of Hb A, respectively. Three of the rHbs, rHb (?L29W), rHb (?L28F), and rHb (?L28W), exhibit very low oxygen affinity and reduced cooperativity compared to Hb A, while the previously studied rHb (?L29F), exhibits high oxygen affinity. In 0.1 M sodium phosphate buffer at pH 7.4 and 29 ºC, the p50 values for rHb (?L29F), rHb (?L29W), rHb (?L28F), rHb (?L28W), and Hb A are 4.0, 32, 36, 68 and 9.3 mm Hg, and the Hill coefficients (nmax) are 2.4, 2.3, 2.6, 2.0, and 3.1, respectively. We have used 1H-NMR to investigate the effects of mutations in the B10 helix of both the ?- and ?-chains on the quaternary structure and the tertiary structure. The ?1?1 and ?1?2 subunit interfaces in both deoxy and liganded states are not perturbed, whereas the tertiary structures around the heme pockets of the mutated chains are perturbed in these four rHbs as expected. The rate constants for the autoxidation and azide-induced oxidation of rHb (?L29F), rHb (?L29W), rHb (?L28F), rHb (?L28W), and Hb A were measured by visual spectrometry. The NO-induced oxidation rates have been determined through experiments using the stopped flow apparatus. The present results provide new insights into the factors that affect the oxygen affinity, cooperativity, and oxidation of hemoglobin.