Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy

Vasily S. Oganesyan, Tim Rasmussen, Shirley Fairhurst, Andrew J. Thomson

Research output: Contribution to journalArticle

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Abstract

The enzyme nitrous oxide reductase (N2OR) has a unique tetranuclear copper centre [Cu4S], called Cu-Z, at the catalytic site for the two-electron reduction of N2O to N-2. The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N2OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu-Z, that undergoes a one-electron redox cycle and Cu-Z*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu-Z showing a rich hyperfine splitting in the g(parallel to)-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu-Z and to calculate EPR parameters. The results for the oxidation state [Cu3+1Cu1+2S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu-Z. The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu-I 20.1%, Cu-II 9.5%, Cu-III 4.8% and Cu-IV 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu-Z, allowing electron redistribution within the plane during interaction with the substrate, N2O.

Original languageEnglish
Pages (from-to)996-1002
Number of pages7
JournalDalton Transactions
Issue number7
Early online date5 Mar 2004
DOIs
Publication statusPublished - 2004

Keywords

  • electron-paramagnetic-resonance
  • density-functional theory
  • Paracoccus-denitrificans
  • N2O reductase
  • Pseudomonas-stutzeri
  • cytochrome-C
  • Z cluster
  • G-tensor
  • approximation
  • mechanism

Cite this

Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy. / Oganesyan, Vasily S. ; Rasmussen, Tim; Fairhurst, Shirley; Thomson, Andrew J.

In: Dalton Transactions, No. 7, 2004, p. 996-1002.

Research output: Contribution to journalArticle

Oganesyan, Vasily S. ; Rasmussen, Tim ; Fairhurst, Shirley ; Thomson, Andrew J. / Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy. In: Dalton Transactions. 2004 ; No. 7. pp. 996-1002.
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abstract = "The enzyme nitrous oxide reductase (N2OR) has a unique tetranuclear copper centre [Cu4S], called Cu-Z, at the catalytic site for the two-electron reduction of N2O to N-2. The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N2OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu-Z, that undergoes a one-electron redox cycle and Cu-Z*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu-Z showing a rich hyperfine splitting in the g(parallel to)-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu-Z and to calculate EPR parameters. The results for the oxidation state [Cu3+1Cu1+2S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu-Z. The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu-I 20.1{\%}, Cu-II 9.5{\%}, Cu-III 4.8{\%} and Cu-IV 9.2{\%}, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8{\%} spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu-Z, allowing electron redistribution within the plane during interaction with the substrate, N2O.",
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T1 - Characterisation of [Cu4S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy

AU - Oganesyan, Vasily S.

AU - Rasmussen, Tim

AU - Fairhurst, Shirley

AU - Thomson, Andrew J.

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N2 - The enzyme nitrous oxide reductase (N2OR) has a unique tetranuclear copper centre [Cu4S], called Cu-Z, at the catalytic site for the two-electron reduction of N2O to N-2. The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N2OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu-Z, that undergoes a one-electron redox cycle and Cu-Z*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu-Z showing a rich hyperfine splitting in the g(parallel to)-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu-Z and to calculate EPR parameters. The results for the oxidation state [Cu3+1Cu1+2S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu-Z. The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu-I 20.1%, Cu-II 9.5%, Cu-III 4.8% and Cu-IV 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu-Z, allowing electron redistribution within the plane during interaction with the substrate, N2O.

AB - The enzyme nitrous oxide reductase (N2OR) has a unique tetranuclear copper centre [Cu4S], called Cu-Z, at the catalytic site for the two-electron reduction of N2O to N-2. The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N2OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu-Z, that undergoes a one-electron redox cycle and Cu-Z*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu-Z showing a rich hyperfine splitting in the g(parallel to)-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu-Z and to calculate EPR parameters. The results for the oxidation state [Cu3+1Cu1+2S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu-Z. The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu-I 20.1%, Cu-II 9.5%, Cu-III 4.8% and Cu-IV 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu-Z, allowing electron redistribution within the plane during interaction with the substrate, N2O.

KW - electron-paramagnetic-resonance

KW - density-functional theory

KW - Paracoccus-denitrificans

KW - N2O reductase

KW - Pseudomonas-stutzeri

KW - cytochrome-C

KW - Z cluster

KW - G-tensor

KW - approximation

KW - mechanism

U2 - 10.1039/b313913a

DO - 10.1039/b313913a

M3 - Article

SP - 996

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JO - Dalton Transactions

JF - Dalton Transactions

SN - 1477-9226

IS - 7

ER -