On the mechanism of action of cytochrome P-450. Spectral intermediates in the reaction with iodosobenzene and its derivatives.
Cytochrome P-450 is known to catalyze the following oxygen transfer reaction: RH + PhIO----ROH + PhI where RH represents a variety of hydroxylatable substrates and PhIO a variety of iodosobenzene derivatives that serve as oxygen donors, and neither molecular oxygen nor an external electron donor is required. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interaction with a variety of substituted iodosobenzenes and iodobenzene diacetates have been determined by stopped flow spectrophotometry. The reaction of phenobarbital-induced rabbit liver microsomal cytochrome P-450 form 2 with iodosobenzenes or iodobenzene diacetates leads to the reversible formation of three spectral intermediates, termed E, F, and G. Complex E is characterized by a type I difference spectrum, representing the iodosobenzene-dependent partial shift of the low spin hexacoordinate form of the ferric enzyme to the high spin pentacoordinate form, F represents a transient intermediate whose spectrum cannot be determined for kinetic reasons, and G represents a blue-shifted intermediate with an absorption maximum at about 393 nm in the absolute spectrum. The striking and principal feature of these observations is that the spectrum of Complex G does not vary with structural differences in the iodosobenzene derivatives, in contrast to the transient species observed in previous studies in this laboratory in the reaction between cytochrome P-450 form 2 and aromatic peroxy compounds. Complex G exhibits the spectral properties one might anticipate for an iron-oxo intermediate containing only one oxygen atom derived from the starting iodosobenzene.
Blake
R C
RC
Department of Biological Chemistry, Medical School, the University of Michigan, Ann Arbor 48109-0606.
Coon
M J
MJ
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology* / Q000648:ultrastructure; D007462:Iodobenzenes / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D000442:Octanols; D011817:Rabbits; D012995:Solubility; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014867:Water
D007462:Iodobenzenes; D000442:Octanols; D014867:Water; D003577:Cytochrome P-450 Enzyme System; C037276:iodosobenzene