Bidirectional catalysis by copper-containing nitrite reductase

HJ Wijma, GW Canters, S de Vries, MP Verbeet*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

52 Citations (Scopus)

Abstract

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K-eq) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K-eq amounts to 103. The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the kat for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1).

Original languageEnglish
Pages (from-to)10467-10474
Number of pages8
JournalBiochemistry
Volume43
Issue number32
DOIs
Publication statusPublished - 17-Aug-2004

Keywords

  • ALCALIGENES-FAECALIS S-6
  • X-RAY-STRUCTURE
  • INTRAMOLECULAR ELECTRON-TRANSFER
  • ACHROMOBACTER-CYCLOCLASTES
  • TYPE-2 COPPER
  • DISSIMILATORY NITRITE
  • ACTIVE-SITE
  • XYLOSOXIDANS NCIMB-11015
  • ANGSTROM RESOLUTION
  • SUBSTRATE-BINDING

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