An O‐Centered Structure of the Trinuclear Copper Center in the Cys500Ser/Glu506Gln Mutant of CueO and Structural Changes in Low to High X‐Ray Dose Conditions

Kakemoto, Hirofumi; Sugiyama, Ryosuke; Kataoka, Keisuke; Higuchi, Yoshiki; Sakurai, Takeshi

doi:10.1002/ange.201107739

Cited by 6 publications

(7 citation statements)

References 32 publications

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“…This can now be done rather routinely at synchrotron sites, especially using online optical microspectrophotometry 20 as well as by X-ray absorption spectroscopy. 28 Protein crystals are typically grown aerobically and thus maintain the oxidized state of their metal ion sites. X-rays have been found to cause reduction of the protein's redox site, notably a metal ion, primarily because this is the thermodynamically favored site of reduction 29 by the primary irradiation produced hydrated electrons and secondary reducing radicals.…”

Section: Ra D I a Ti On In D U Ce D C Ha N Ge S Wi Th In R Ed Ox P mentioning

confidence: 99%

Radiation chemists look at damage in redox proteins induced by X‐rays

Wherland

Pecht

2018

Proteins

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The three-dimensional structure of proteins, especially as determined by X-ray crystallography, is critical to the understanding of their function. However, the X-ray exposure may lead to damage that must be recognized and understood to interpret the crystallographic results. This is especially relevant for proteins with transition metal ions that can be oxidized or reduced. The detailed study of proteins in aqueous solution by the technique of pulse radiolysis has provided a wealth of information on the production and fate of radicals that are the same as those produced by X-ray exposure. The results reviewed here illustrate how the products of the interaction of radiation with water or with solutes added to the crystallization medium, and with proteins themselves, are formed, and about their fate. Of particular focus is how electrons are produced and transferred through the polypeptide matrix to redox centers such as metal ions or to specific amino acid residues, for example, disulfides, and how the hydroxyl radicals formed may be converted to reducing equivalents or scavenged.

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Section: Ra D I a Ti On In D U Ce D C Ha N Ge S Wi Th In R Ed Ox P mentioning

confidence: 99%

Radiation chemists look at damage in redox proteins induced by X‐rays

Wherland

Pecht

2018

Proteins

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“…A recent crystallographic study on a double mutant Cys500Ser/Glu506Gln showed that the hydrogen bond network leading from the exterior of the protein molecule to TNC formed with the amide group in the side chain of the Gln residue [14]. However, the carboxyl group of the side chain of Glu is distinct from the amide group in the side chain of Gln which does not cycle between the protonated and deprotonated forms [7].…”

Section: Reduction Of O 2 To 2h 2 Omentioning

confidence: 99%

“…The mutations of Glu506 with Asp, Ala, and Ile were performed to impose a variety perturbations on the hydrogen bond network, and to compare the results with data obtained from the previous Gln mutation which inactivates the hydrogen bond network with respect to proton transfer [6,7,14]. We expect that the hydrogen bond network is modified, but still highly functional in the Glu506Asp mutant.…”

Section: Introductionmentioning

confidence: 99%

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

Kajikawa

Kataoka

Sakurai

2012

Biochemical and Biophysical Research Communications

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We have the pleasure for submitting our manuscript entitled "Modifications on the Hydrogen Bond Network by Mutations of Escherichia coli CueO Affect the Process of Proton Transfer to Dioxygen Leading to Peculiar Alterations of Enzymatic Activities" inBiochemical Biophysical Research Communications. CueO is the cuprous oxidase from Escherichia coli and concerns in a system to excrete excess copper to protect E. coli from oxidative damages. Together with laccase and related enzymes, CueO is a multiocopper oxidase and has the type I copper to mediate the electron transfer from substrate and the trinuclear copper center comprised of type II copper and a pair of type III coppers, at which dioxygen is four-electron reduced without forming activated oxygen species. This conversion of dioxygen to water molecule is performed only by terminal oxidases and multicopper oxidases. Therefore, multicopper oxidases are expected to apply to cathodic enzyme for biofuel cells. We have trapped reaction intermediates by preparing mutants and derivatives of multicopper oxidases, which are electron deficient to fully reduce dioxygen and are difficult to relay protons to dioxygen.We revealed that Glu506 in CueO located adjacent to the trinuclear copper center is a key amino acid to relay protons to dioxygen, because this amino is located in the hydrogen bond network leading from the exterior of the CueO molecule to the trinuclear copper center. We performed systematic mutations at Glu506 and Asp507 to study proton relay mechanism in the course of the four-electron reduction of dioxygen by multicopper oxidases. We found that the conserved Glu506 is exchangeable with Asp.The Ala mutation was also functional presumably because one or two water molecules occupied the open space and the alternative proton relay network was constructed in contrast to the Ala mutations in cytochrome oxidases. The hydrogen bond network was also constructed by the Gln mutation, but Gln had not ability to relay protons because the amide group do not cycle between protonated and deprotonated forms. Cover LetterThe Ile mutation resulted in complete loss of enzymatic activity because an open space to accommodate a water molecule is not formed due to bulkiness in the side chain.Thus the present systematic mutations at Glu506 and also Asp507, which is located in a branch of the hydrogen bond network and was found to play a role to support the proton transfer, unequivocally showed clear results of the proton relay pathway in multicopper oxidase for the first time. Due to special attention to the four-electron reduction of dixoygen by multicopper oxidases, we believe the present study attracts wide attention from researches studying on biochemistry, inorganic chemistry, electrochemistry and related fields, and this is the reason why we would like to publish the present study as rapidly as possible in BBRC.Proton transfer pathway to dioxygen in CueO was identified.Glu506 is the key amino acid to transport proton.The Ala mutation at Glu506 formed a compensatory proton tra...

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“…1) [11,[19][20][21]. Cycling of protonation and deprotonation in the side chain of the conserved Glu residue coupled with the change in redox state of BOD and CueO has been observed by FT-IR studies [22].…”

Section: Introductionmentioning

confidence: 99%

“…Cycling of protonation and deprotonation in the side chain of the conserved Glu residue coupled with the change in redox state of BOD and CueO has been observed by FT-IR studies [22]. 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

Kataoka

et al. 2014

Biochemical and Biophysical Research Communications

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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.

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An O‐Centered Structure of the Trinuclear Copper Center in the Cys500Ser/Glu506Gln Mutant of CueO and Structural Changes in Low to High X‐Ray Dose Conditions

Cited by 6 publications

References 32 publications

Radiation chemists look at damage in redox proteins induced by X‐rays

Radiation chemists look at damage in redox proteins induced by X‐rays

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

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

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