TY - JOUR
T1 - Roles of Proximal Ligand in Heme Proteins
T2 - Replacement of Proximal Histidine of Human Myoglobin with Cysteine and Tyrosine by Site-Directed Mutagenesis as Models for P-450, Chloroperoxidase, and Catalase
AU - Adachi, Shin Ichi
AU - Nagano, Shingo
AU - Ishimori, Koichiro
AU - Watanabe, Yoshihito
AU - Morishima, Isao
AU - Egawa, Tsuyoshi
AU - Kitagawa, Teizo
AU - Makino, Ryu
PY - 1993/1/1
Y1 - 1993/1/1
N2 - Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted 1H-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O–O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O–O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
AB - Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted 1H-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O–O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O–O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
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U2 - 10.1021/bi00052a031
DO - 10.1021/bi00052a031
M3 - Article
C2 - 8380334
AN - SCOPUS:0027505784
SN - 0006-2960
VL - 32
SP - 241
EP - 252
JO - Biochemistry
JF - Biochemistry
IS - 1
ER -