P450 electron transfer mechanics: unravelling the evolutionary role of transient redox protein complexes

Abstract The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains amystery in biology. It is widely believed that sterol 14alpha-demethylase (CYP51), an essential enzyme ofsterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species indifferent biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamicssimulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redoxpartner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterolproducing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanicsin the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules invarious orientations and the interactions are transient. Close proximity of ferredoxin, however, is requiredto complete the substrate-induced large-scale structural switch in the P450 domain that enables protoncoupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamentalimplications regarding the early evolution of electron transfer proteins and for the redox reactions in theearly steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequentdiversification of the P450 electron transfer machinery in nature.