科研之友
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摘要
The E11 valine in the distal heme pocket of either the alpha- or beta-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. H-1 NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the alpha(1)beta(2) interface in either form, whereas the H-bond between alpha His-103 and beta-Gln-131 in the alpha(1)beta(1) interfaces of the deoxy alpha-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (alpha V62F), rHb (beta V67I), and rHb (beta V67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the beta-subunit of rHb (beta V67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (alpha V62I), rHb (alpha V62F), and rHb (beta V67F). Functionally, the oxygen binding affinity (P-50), cooperativity (n(50)), and the alkaline Bohr Effect of the three alpha-subunit mutants and rHb (beta V67L) are similar to those of Hb A. rHb (beta V67I) and rHb (beta V67F) exhibit low and high oxygen affinity, respectively. rHb (beta V67F) has P-50 values lower that those reported for rHb (alpha L29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.
- 出版日期2013-8-30
