Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

Journal of Inorganic Biochemistry

et al. 2015

Self Cite

A multicopper oxidase, CueO was doubly mutated at its type I copper ligand, Cys500 and an acidic amino acid residue located in the proton transfer pathway, Glu506, to Ser and Ala, respectively.Cys500Ser/Glu506Ala was mainly in a novel resting form to afford the absorption band at ca. 400 nm and an EPR signal with a highly anisotropic character derived from type III copper. However, Cys500Ser/Glu506Ala gave the same reaction intermediate (peroxide intermediate) as that from Cys500Ser and Cys500Ser/Glu506Glu. with O 2 because one electron is deficient to form two water molecules [5]. On the other hand, the hydrogen bond network constructed with a conserved Glu residue (Glu506) and water molecules has been proved to function as a proton transport pathway from bulk water to TNC [6,8.9,[12][13][14] (Fig. 1). A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Mutations of this A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT4 the mutation, constructing a compensatory hydrogen bond network with only water molecules [9].In the present study we report unique spectral and magnetic properties of a double mutant of CueO, Cys500Ser/Glu506Ala at a ligand to T1Cu, Cys500 and at Glu506. The reaction of the double mutant with O 2 has also been performed to study properties of the intermediate I in comparison with that formed from Cys500Ser and Cys500Ser/Glu506Gln.Cys500Ser/Glu506Ala contained 3.2 Cu atoms in a protein molecule as determined from atomic absorption spectroscopy. Due to the absence of T1Cu the absorption bands at 600 nm disappeared, although the d-d band from TNC ( 745 ~400 M -1 cm -1 ) was observable ( Fig. 2A, full line).The band at 330 nm was considerably weakened ( ~2000 M -1 cm -1 relative to ~5200 M -1 cm -1 for the wild type enzyme (dotted line)), but a novel shoulder band was unequivocally observed at ca. 400 nm.This absorption band disappeared upon reduction, indicating that it did not come from an impurity.The corresponding circular dichrosim (CD) spectrum (Fig. 2B, full line) afforded a negatively signed band at 400 nm in addition to the weakened negatively signed band at 330 nm and the d-d bands at ca. 700 nm and 860 nm which were inverted in sign from those of Cys500Ser (dashed line). The EPR spectrum of Cys500Ser/Glu506Ala (Fig. 2C, full line) afforded a rhombic Cu(II) signal (g z = 2.40, g y = 2.11, g x = 2.04, A z = 7.6 x 10 -3 cm -1 , and A x = 5.3 x 10 -3 cm -1 ) differing from the resting CueO and Cys500Ser ( Fig. 2C dashed line). The EPR signal of T2Cu (g II = 2.25, A II = 19.6 x 10 -3 cm -1 ) was apparently minor. The total EPR-detectable Cu(II) in Cys500Ser/Glu500Ala was 2.2 per a protein molecule. Therefore, Cys500Ser/Glu506Ala is supposed to be in a unique resting state, both T3Cus to be magnetically isolated and T2Cu to be cuprous. Cys500Ser/Glu500Gln. Freezing of Cys500Ser/Glu506Ala soon after the reaction with O 2 gave the weak T2Cu EPR signal ( Supplementary Fig. 1, The total EPR-detectable Cu(II) was ~0.2 per a protein molecule), indicating that the main species ...

Section: Accepted Manuscriptmentioning

confidence: 99%

“…Cu 2+ ion has never been incorporated into the T1Cu site, of which Cys ligand is changed to Ser [2,[5][6][7][8][9].…”

Section: Based On Spectral Property and Enzymatic Reactivity The Restmentioning

confidence: 99%

A novel resting form of the trinuclear copper center in the double mutant of a multicopper oxidase, CueO, Cys500Ser/Glu506Ala

Kajikawa

Sugiyama

Journal of Inorganic Biochemistry

et al. 2015

Self Cite

“…Averaged Cu contents in the single and double mutants were 4 and 3, respectively, within the experimental error of 10% (Table 1). The single mutants, Glu463Gln and Glu463Ala exhibited very low enzymatic activities, although the corresponding Ala mutants of CueO and Fet3p showed considerably high enzymatic activities [19,23]. On the other hand, the double mutants did not show enzymatic activities due to the mutation at the T1Cu site [12].…”

Section: Characterizations Of Mutantsmentioning

confidence: 99%

“…Mutations on the conserved Glu residue have been performed for CueO and Fe3p, resulting in the successful trapping and characterization of the intermediate II [11,19,21]. Mutations of this Glu residue with Gln led to practically complete loss in enzymatic activities, but its substitution with Ala exhibited considerably high enzymatic activities due to a formation of compensatory hydrogen bond network with only water molecules [19,23,24], while it was found that Asp located in one of other two channels leading to TNC also concerns profoundly in the formation of water molecules [25,26].…”

Section: Introductionmentioning

confidence: 99%

Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

Morishita

Kurita

Biochemical and Biophysical Research Communications

et al. 2014

Self Cite

Bilirubin oxidase is a multicopper oxidase containing a type I Cu center to specifically oxidize bilirubin and related substrates and a trinuclear Cu center to perform four-electron reduction of dioxygen as the final electron acceptor. Special attentions have been paid on multicopper oxidases, since this class of enzymes has potential uses as electrocatalyst for biofuel cells and biosensor and catalyst to form a variety of dyes. In addition, bilirubin oxidase has been utilized for the clinical test of liver. In spite of wide attention and potential wide use of the enzyme, structural and functional studies on bilirubin oxidase have been limited.The present study is on the proton transfer mechanism for dioxygen reduction by bilirubin oxidase. We performed mutation at Glu463 located in the hydrogen bond network leading from bulk water to the active site deeply buried inside protein molecule. By singly performing mutation at this amino acid and doubly performing mutation at a Cys ligand to type I Cu center in addition to the mutation at Glu463, we could trap two reaction intermediates, and characterized them in comparisons with those trapped in other multicopper oxidases.We believe this study attracts wide attention from researches studying on biochemistry, bioinorganic chemistry, electrochemistry, biophysics and related fields including bioinspired chemistry, and contributes in the understanding of the four-electron reduction by multicopper oxidases and more widely, transport of protons in protein molecules. According to reasons as above we would like to publish our results in the world wide BBRC. sincerely yours, Takeshi Sakurai Cover LetterProton transport pathway in bilirubin oxidase was mutated Two intermediates in the dioxygen reduction steps were trapped and characterized.A specific glutamate for dioxygen reduction by multicopper oxidases was identified.

“…presents unequivocal evidence that the hydrogen bond between Asp439 and His443 functions as an electron-transfer pathway. 8,15 Nevertheless, the hydrogen bond is not the exclusive electron-transfer pathway, as judged from the fact that the copper(I) oxidase activity has been reserved in the Asp439Ala mutants by ca. 50% from those of the parent enzymes.…”

mentioning

confidence: 99%

Role of Hydrogen Bond Connecting Ligands for Substrate and Type I Copper in Copper(I) Oxidase CueO

Sakurai

2013

Chemistry Letters

Self Cite

Mutational and kinetic studies on Asp439, a ligand for the substrate-binding site in the copper(I) oxidase—CueO—and the deletion mutant—Δα5–7 CueO—indicated that the electron transfer from the copper(I) ion as the substrate to the type I copper is performed though the hydrogen bond between Asp439 and His443, a ligand for the type I copper, and also by means of the through-space mechanism directly.