Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy

NADPH-cytochrome P450 oxidoreductase (CYPOR) was shown to undergo large conformational rearrangements in its functional cycle. Using a new Förster resonance energy transfer (FRET) approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor–acceptor distance distribution in the reduced and oxidized states of CYPOR. The unmatched time resolution of TA allowed the quantitative assessment of the donor–acceptor FRET, indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner. The described ultrafast TA measurements of FRET with readily available red–infrared fluorescent labels open new opportunities for structural studies in chromophore-rich proteins and their complexes