Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center.
The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys(500), a type I copper ligand, and the noncoordinating Asp(112) and Glu(506), which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Deltaalpha5-7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu(506) involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp(112) and Glu(506) are discussed.
Kataoka
Kunishige
K
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
Sugiyama
Ryosuke
R
Hirota
Shun
S
Inoue
Megumi
M
Urata
Kanae
K
Minagawa
Yoichi
Y
Seo
Daisuke
D
Sakurai
Takeshi
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D003300:Copper; D010088:Oxidoreductases; C499679:cueO protein, E coli; D010100:Oxygen