Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam

Shingo Nagano, Hideo Shimada, Akiko Tarumi, Takako Hishiki, Yoko Kimata-Ariga, Tsuyoshi Egawa, Makoto Suematsu, Sam Yong Park, Shin Ichi Adachi, Yoshitsugu Shiro, Yuzuru Ishimura

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm-1. The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm-1 band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O 2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

Original languageEnglish (US)
Pages (from-to)14507-14514
Number of pages8
JournalBiochemistry
Volume42
Issue number49
DOIs
StatePublished - Dec 16 2003
Externally publishedYes

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Camphor 5-Monooxygenase
Cytochromes
Infrared radiation
Carbon Monoxide
Electrons
Hydroxylation
putidaredoxin
Heme
Stretching
Catalyst activity
Catalytic Domain
Iron
Crystal structure
Oxygen
Ligands
Amino Acids
Mutation
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., ... Ishimura, Y. (2003). Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam. Biochemistry, 42(49), 14507-14514. https://doi.org/10.1021/bi035410p

Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam. / Nagano, Shingo; Shimada, Hideo; Tarumi, Akiko; Hishiki, Takako; Kimata-Ariga, Yoko; Egawa, Tsuyoshi; Suematsu, Makoto; Park, Sam Yong; Adachi, Shin Ichi; Shiro, Yoshitsugu; Ishimura, Yuzuru.

In: Biochemistry, Vol. 42, No. 49, 16.12.2003, p. 14507-14514.

Research output: Contribution to journalArticle

Nagano, S, Shimada, H, Tarumi, A, Hishiki, T, Kimata-Ariga, Y, Egawa, T, Suematsu, M, Park, SY, Adachi, SI, Shiro, Y & Ishimura, Y 2003, 'Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam', Biochemistry, vol. 42, no. 49, pp. 14507-14514. https://doi.org/10.1021/bi035410p
Nagano S, Shimada H, Tarumi A, Hishiki T, Kimata-Ariga Y, Egawa T et al. Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam. Biochemistry. 2003 Dec 16;42(49):14507-14514. https://doi.org/10.1021/bi035410p
Nagano, Shingo ; Shimada, Hideo ; Tarumi, Akiko ; Hishiki, Takako ; Kimata-Ariga, Yoko ; Egawa, Tsuyoshi ; Suematsu, Makoto ; Park, Sam Yong ; Adachi, Shin Ichi ; Shiro, Yoshitsugu ; Ishimura, Yuzuru. / Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam. In: Biochemistry. 2003 ; Vol. 42, No. 49. pp. 14507-14514.
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abstract = "Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm-1. The former band is dominant (>95{\%} in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95{\%} in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm-1 band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O 2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.",
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AB - Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm-1. The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm-1 band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O 2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

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