Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin

Hideo Koga, Yasuhiro Sagara, Tsuyoshi Yaoi, Mitsushi Tsujimura, Kazuhide Nakamura, Kazuhisa Sekimizu, Ryu Makino, Hideo Shimada, Yuzuru Ishimura, Kei Yura, Mitiko Go, Masamichi Ikeguchi, Tadao Horiuchi

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60 Citations (Scopus)

Abstract

Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1 400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Original languageEnglish
Pages (from-to)109-113
Number of pages5
JournalFEBS Letters
Volume331
Issue number1-2
DOIs
Publication statusPublished - 1993 Sep 27
Externally publishedYes

Keywords

  • Amino acid substitution
  • Binding of P450cam with putidaredoxin
  • Cytochrome P450cam
  • Electron transfer
  • Putidaredoxin
  • Random mutagenesis

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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    Koga, H., Sagara, Y., Yaoi, T., Tsujimura, M., Nakamura, K., Sekimizu, K., Makino, R., Shimada, H., Ishimura, Y., Yura, K., Go, M., Ikeguchi, M., & Horiuchi, T. (1993). Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin. FEBS Letters, 331(1-2), 109-113. https://doi.org/10.1016/0014-5793(93)80307-G