Cryotrapped reaction intermediates of cytochrome p450 studied by radiolytic reduction with phosphorus-32.
Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using (60)Co gamma-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe(3+)-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999) J. Am. Chem. Soc. 121, 10654-10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at approximately 195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed.
Denisov
I G
IG
Department of Biochemistry, Center for Biophysics and Computational Biology, and Beckman Institute, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
Makris
T M
TM
Sligar
S G
SG
D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D011854:Radiochemistry
D010761:Phosphorus Radioisotopes; D003577:Cytochrome P-450 Enzyme System