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 ¹²CNBu/ ¹³CNBu derivatives. For P450cam, but not P450nor, other ¹³C 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 ¹²C/¹³C 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 ¹³C 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.