Conformational Motion of Ferredoxin Enables Efficient Electron Transfer to Heme in the Full-Length P450TT

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts used in natural products biosynthesis, xenobiotic metabolisms, and biotechnologies. In P450s, the electrons required for O-2 activation are supplied by NAD(P)H through stepwise electron transfers (ETs) mediated by redox partners. While much is known about the machinery of the catalytic cycle of P450s, the mechanisms of long-range ET are largely unknown. Very recently, the first crystal structure of full-length P450(TT) was solved. This enables us to decipher the interdomain ET mechanism between the [2Fe-2S]-containing ferredoxin and the heme, by use of molecular dynamics simulations. In contrast to the distal conformation characterized in the crystal structure where the [2Fe-2S] cluster is similar to 28 angstrom away from heme-Fe, our simulations demonstrated a proximal conformation of [2Fe-2S] that is similar to 17 angstrom [and 13.7 A edge-to-edge] away from heme-Fe, which may enable the interdomain ET. Key residues involved in ET pathways and interdomain complexation were identified, some of which have already been verified by recent mutation studies. The conformational transit of ferredoxin between distal and proximal was found to be controlled mostly by the long-range electrostatic interactions between the ferredoxin domain and the other two domains. Furthermore, our simulations show that the full-length P450(TT) utilizes a flexible ET pathway that resembles either P450(Scc) or P450(cam). Thus, this study provides a uniform picture of the ET process between reductase domains and heme domain.

Automated summary

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts used in various fields, and recent research has revealed new insights into the long-range electron transfer mechanisms within the enzyme. The study utilized molecular dynamics simulations to uncover the interdomain electron transfer process between ferredoxin and heme, providing a clearer understanding of the catalytic cycle of P450s. Additionally, key residues involved in electron transfer pathways were identified, aiding in the development of more efficient biocatalysts in the future.