Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli: Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and MET-95 mutants

Sue Taguchi, Toshitaka Matsui, Jotaro Igarashi, Yukie Sasakura, Yasuyuki Araki, Osamu Ito, Shunpei Sugiyama, Ikuko Sagami, Toru Shimizu

Research output: Contribution to journalArticlepeer-review

48 Citations (Scopus)

Abstract

The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis. The rate of O2 association (kon) with full-length Ec DOS is extremely slow at 0. 0019 μM-1 s-1, compared with >9.5 μM -1 s-1 for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method. This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain. Dissociation constants (Kd) calculated from the kinetic parameters are 340 and 20 μM for the full-length wild-type enzyme and its isolated PAS domain, respectively. Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the kon value by more than 30-fold, and consequently, a decrease in the Kd value to less than 1 Μ. The kon value for CO binding to the full-length wild-type enzyme is also very low (0.00081 μM-1 s -1). The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O2. However, the K d values for CO are considerably lower than those for O2.

Original languageEnglish
Pages (from-to)3340-3347
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number5
DOIs
Publication statusPublished - 2004 Jan 30

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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