Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states.

Electron transfer between membrane spanning oxidoreductase enzymes controls vital metabolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analysis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date has remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR, we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stability by ∼2-fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions, highlighting the importance of studying POR function in a membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule resolution.