Formation of a cytochrome c-nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus

Tim Rasmussen, Thomas Brittain, Ben C Berks, Nicholas J Watmough, Andrew J Thomson

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N-2). N2OR contains two metal centers; a binuclear copper center, Cu-A, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, Cu-z, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer ( k(obs) = 150 s(-1)) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rate of electron transfer was recorded for the reverse reaction, followed by a much slower internal electron transfer from Cu-z to Cu-A (k(obs) = 0.1 - 0.4 s(-1)). The internal electron transfer process is likely to represent the rate-determining step in the catalytic cycle. Remarkably, in the absence of cytochrome c, fully reduced N2OR is inert towards its substrate, even though sufficient electrons are stored to initiate a single turnover. However, in the presence of reduced cytochrome c and N2O, a single turnover occurs after a lag-phase. We propose that a conformational change in N2OR is induced by its specific interaction with cytochrome c that in turn either permits electron transfer between Cu-A and Cu-z or controls the rate of N2O decomposition at the active site.

Original languageEnglish
Pages (from-to)3501-3506
Number of pages6
JournalDalton Transactions
Issue number21
DOIs
Publication statusPublished - 2005

Keywords

  • electron-transfer pathway
  • wolinella-succinogenes
  • denitrifying bacteria
  • multicopper enzyme
  • kinetic-analysis
  • catalytic center
  • denitrificans
  • purification
  • cluster
  • involvement

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