Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli: Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials

Satoshi Hirata, Toshitaka Matsui, Yukie Sasakura, Shunpei Sugiyama, Tokiko Yoshimura, Ikuko Sagami, Toru Shimizu

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.

Original languageEnglish
Pages (from-to)4771-4779
Number of pages9
JournalEuropean Journal of Biochemistry
Volume270
Issue number23
DOIs
Publication statusPublished - 2003 Dec

Keywords

  • Heme sensor
  • Magnetic circular dichroism
  • Optical absorption
  • Phosphodiesterase
  • Redox potential

ASJC Scopus subject areas

  • Biochemistry

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