Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd1 nitrite reductase: Regulation of the NO-bound end product

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Abstract

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome c553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c552red→NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1±0.1×105 M-1 s-1 (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s-1 could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme cred→NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme cred→NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4±0.7 s-1, corresponding to a k2 value of 2.2±1.1×104 M-1 s-1 for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.

Original languageEnglish
Pages (from-to)55-62
Number of pages8
JournalJournal of Biological Inorganic Chemistry
Volume6
Issue number1
DOIs
Publication statusPublished - 17 Sep 2001

Keywords

  • Catalytic mechanism
  • Cytochrome cd
  • Electrochemistry
  • Electron transfer
  • Nitrite reductase

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