TY - JOUR
T1 - Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin. Site-directed mutagenesis of the distal histidine
AU - Springer, B. A.
AU - Egeberg, K. D.
AU - Sligar, S. G.
AU - Rohlfs, R. J.
AU - Mathews, A. J.
AU - Olson, J. S.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1989
Y1 - 1989
N2 - Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64 → Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64 → Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were ~50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were ~5-15-fold higher than those for the native protein. All mutants studied (except His-64 → Tyr) showed ~10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-300-fold greater K (K(CO)/K(O2)) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.
AB - Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64 → Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64 → Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were ~50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were ~5-15-fold higher than those for the native protein. All mutants studied (except His-64 → Tyr) showed ~10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-300-fold greater K (K(CO)/K(O2)) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.
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M3 - Article
C2 - 2644279
AN - SCOPUS:0024519403
VL - 264
SP - 3057
EP - 3060
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 6
ER -