Roles of proximal ligand in heme proteins: replacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase, and catalase.
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.
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
S
S
Adachi
S
S
Nagano
K
K
Ishimori
Y
Y
Watanabe
I
I
Morishima
T
T
Egawa
T
T
Kitagawa
R
R
Makino
D001555:Benzene Derivatives / Q000378:metabolism; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry; D002711:Chloride Peroxidase / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D001555:Benzene Derivatives; D009211:Myoglobin; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D002711:Chloride Peroxidase; D002374:Catalase; D003545:Cysteine; C007164:cumene hydroperoxide