Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.
Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.
Tomita
T
T
Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki 444-8585, Japan.
Ogo
S
S
Egawa
T
T
Shimada
H
H
Okamoto
N
N
Imai
Y
Y
Watanabe
Y
Y
Ishimura
Y
Y
Kitagawa
T
T
D000818:Animals; D003486:Cyanides / Q000032:analysis* / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007259:Infrared Rays; D008861:Microsomes / Q000737:chemistry; D008862:Microsomes, Liver / Q000737:chemistry; D009570:Nitriles / Q000737:chemistry; D011817:Rabbits; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013997:Time Factors
D003486:Cyanides; D009570:Nitriles; C002990:butyl isocyanide