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 redu ction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the cat alytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Usin g Co-60 gamma -irradiation, the primary reduced oxy-P450 species at 77 K ha s been identified as a superoxo- or hydroperoxo-Fe3+-heme complex (Davydov, R., Macdonald, I. D, G,, Makris, T, M., Sligar, S. G., and Hoffman, B, M. (1999) J, Am. Chem. Sec. 121, 10654-10655). The electronic absorption spect roscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagne tic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. me determine the W-visible optical spectra of the reduced ox ygenated state of CYP101 and show that the primary intermediate, a hydroper oxo-P450, is stable below 180 K and converts smoothly to the product comple x at similar to 195 K, In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compoun d I type spectrum) was observed.