Recombinant human hemoglobin: modification of the polarity of the beta-heme pocket by a valine67(E11)-->threonine mutation.
Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)
Fronticelli
C
C
Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.
Brinigar
W S
WS
Olson
J S
JS
Bucci
E
E
Gryczynski
Z
Z
O'Donnell
J K
JK
Kowalczyk
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014633:Valine
D006454:Hemoglobins; D008024:Ligands; D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D013912:Threonine; D006418:Heme; D014633:Valine