Electron transfer between membrane spanning oxi-doreductase enzymes crucially controls vital meta-bolic 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 analy-sis 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 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 stabil-ity by ~2 fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions high-lighting the importance of studying POR function in membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule reso-lution.
Acs Chemical Biology, 2014, Vol 9, Issue 3, p. 630-634