Structural Studies of the Docking Complex between Cytochrome P450 (CYP101) and Putidaredoxin (PDX) by Double Electron-Electron Resonance (DEER)

Abstract

P450cam (CYP101) is an archetype of the cytochrome P450 superfamily of heme mono-oxygenases. This enzyme hydroxylates camphor to 5-exo-hydroxy-camphor following two distinct electron transfers from its unique [2Fe-2S] redox partner, Pdx (Putidaredoxin). Other ferredoxin-type proteins, such as adrenodoxin, are only able transfer the first electron and lack the features of the complex of P450:Pdx required for the second electron transfer. This specificity of the protein electron donor suggests that geometry of the protein-protein complex may play a critical role in the electron transfer process. Moreover, we have observed in previous studies that P450cam shifts from open to closed conformation upon binding camphor, and that oxidized but not reduced Pdx induces a shift of substrate-bound P450cam from the closed to the open conformation. The above results lead us to pursue structural information about the solution complex of P450cam and Pdx in different redox and coordination states. Several combinations of site-directed spin labeling (SDSL) sites were made with single methanethiosulfonate (MTSL) spin-labels on P450 and Pdx. The inter-spin label distance constraints in the P450:Pdx complex were measured by Double Electron Electron Resonance (DEER or PELDOR). While these studies show that the solution complex between P450cam and Pdx is consistent with that reported in recent crystallographic studies, the studies also show little difference in the docking conformation of P450cam(oxidized)-Pdx(oxidized) and P450cam(reduced CO)-Pdx(reduced). We will also report DEER studies of the conformational states of P450cam bound to CN- and NO. These results will help clarify the structural factors responsible for the role of Pdx as a structural effector of P450cam function.