Denaturation of cytochrome P450 2B1 by guanidine hydrochloride and urea: evidence for a metastable intermediate state of the active site.

Abstract

A metastable intermediate was found in the course of the denaturation of purified cytochrome P450 2B1 by increasing concentrations of guanidine hydrochloride (GuHCl). The metastable intermediate has no or low absorbance at 450 nm in the reduced carbon monoxide difference spectrum and has no absorbance at 420 nm. The intermediate is easily converted to P420 by increasing concentrations of GuHCl. Before it becomes P420, the cytochrome can be completely reconverted to native P450 by dilution and incubation at 4 degrees C. Cytochrome P420 resulting from exposure to higher concentrations of GuHCl (> 3 M) failed to be reconverted to P450 by dilution. Denaturation of P450 2B1 by exposure to low concentrations of urea (< 2 M) is also completely reversible but no obvious intermediate is detectable. An intermediate is observed, however, when the urea denaturation is conducted in the presence of 1 M NaCl. As is the case with higher concentrations of GuHCl, cytochrome P450 denatured by exposure to 5 M or higher concentrations of urea is not reversible. The failure of reconversion of P420 denatured by exposure of cytochrome P450 to high concentrations of GuHCl or urea is probably attributable to the extensive unfolding of the apoprotein, which favors aggregation, rather than to heme loss. Our results also suggest that the active site is more sensitive to denaturants than other regions of the protein.