Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy.

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by γ-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; 14N, 1H, hyperfine couplings). It also includes dramatic changes in reactivity:  the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of ...