The single turnover of 1R(+)camphor-bound oxyferrous cytochrome P450 CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 °C by double-mixing rapid-scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting vs. reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high- and low-spin Fe(III), and high-spin ferric camphor-bound enzyme. The rates of the first two steps, ~140 s-1 and ~85 s-1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low-spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a pre-formed reduced P450-CAM·Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state, and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e., during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first, but not the second electron.