Double electron-electron resonance (DEER) spectroscopy was used to determine the conformational state in solution for the heme monooxygenase P450cam when bound to its natural redox partner, putidaredoxin (Pdx). When oxidized Pdx was titrated into substrate-bound ferric P450cam, the enzyme shifted from the closed to the open conformation. In sharp contrast, however, the enzyme remained in the closed conformation when ferrous-CO P450cam was titrated with reduced Pdx. This result fully supports the proposal that binding of oxidized Pdx to P450cam opposes the open-to-closed transition induced by substrate binding. However, the data strongly suggest that in solution, binding of reduced Pdx to P450cam does not favor the open conformation. This supports a model in which substrate recognition is associated with the open-to-closed transition and electron transfer from Pdx occurs in the closed conformation. The opening of the enzyme in the ferric-hydroperoxo state following electron transfer from Pdx would provide for efficient O-2 bond activation, substrate oxidation, and product release.