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

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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.

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

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S. Y., Adachi, S. I., Shiro, Y., & 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