Preparation and reactions of myoglobin mutants bearing both proximal cysteine ligand and hydrophobic distal cavity: protein models for the active site of P-450.
We have reported that H93C human myoglobin (Mb), in which proximal histidine (His93, F8) was replaced by cysteine, gave nearly identical spectroscopic features of P-450 [Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T., & Makino R. (1993) Biochemistry 32, 241-252]. More importantly, the thiolate ligand enhanced its oxygenation activities when supported by H2O2 due to the exclusive encouragement of heterolytic O-O bond cleavage of peroxides. While we have attributed the enhanced heterolysis to the electron donation from the thiolate ligand, possible participation of the distal histidine (H64, E7) in H93C Mb cannot be eliminated. In addition, the racemic product formation catalyzed by H93C Mb implied that its distal cavity could prevent substrates from accessing to the heme and the reactions may proceed other than by the P-450 type mechanism (ferryl oxygen transfer). In order to clarify whether the distal histidine is involved in the O-O bond cleavage step and to improve accessibility of substrates, the distal histidine of H93C Mb is replaced by smaller and nonpolar residues, glycine (H64G/H93C Mb) and valine (H64V/H93C Mb), by site-directed mutagenesis. Various spectroscopic studies on these double-mutated Mbs revealed the ligation of cysteine to the ferric heme as a thiolate form. In the reaction with cumene hydroperoxide, the anionic nature of the proximal cysteine in H64G/H93C and H64V/H93C Mbs was found to encourage the heterolytic O-O bond cleavage as observed for H93C Mb. The results clearly demonstrate that the distal histidine of H93C Mb is hardly involved in the O-O bond cleavage step and are in good agreement with the role of thiolate ligation for the formation of the reactive intermediate, equivalent to compound I, in the catalytic cycle of P-450 reactions. In the oxygenation of methyl p-tolyl sulfide, the ratios of ferryl oxygen transfer increased in H64G/H93C Mb (58%) and H64V/H93C Mb (78%) as compared to H93C Mb (53%). The increased ratios of ferryl oxygen transfer imply the active site of H64G/H93C and H64V/H93C Mbs being more accessible for substrates; however, the sulfoxidation by the ferric mutant Mbs/H2O2 system was much slower than that by H93C Mb. The poor activities of these mutant Mbs are attributed to the significantly discouraged binding of H2O2.
Matsui
T
T
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Nagano
S
S
Ishimori
K
K
Watanabe
Y
Y
Morishima
I
I
D000098:Acetophenones / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013454:Sulfoxides / Q000378:metabolism
D000098:Acetophenones; D000814:Aniline Compounds; D001555:Benzene Derivatives; D006420:Hemeproteins; D009211:Myoglobin; D011994:Recombinant Proteins; D013454:Sulfoxides; D006418:Heme; D006639:Histidine; C015157:N,N-dimethylaniline; D003577:Cytochrome P-450 Enzyme System; D003545:Cysteine; C007164:cumene hydroperoxide; C038699:acetophenone