Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy

SIMPLE SUMMARY: NADPH-cytochrome P450 reductase (CPR) enzymes are known to undergo an ample conformational transition between the closed and open states in the process of their redox cycling. To explore the conformational landscape of CPR from the potential biofuel crop Sorghum bicolor (SbCPR), we incorporated a FRET donor/acceptor pair into the enzyme and employed rapid scanning stop-flow and pressure perturbation spectroscopy to characterize the equilibrium between its open and closed states at different stages of the redox cycle. Our results suggest the presence of several open conformational sub-states differing in the system volume change associated with the opening transition (ΔV(0)). Although the closed conformation always predominates in the conformational landscape, the population of the open conformations increases by order of magnitude upon the two-electron reduction and the formation of the disemiquinone state of the enzyme. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions. ABSTRACT: NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV(0)) of the opening transition. While the ΔV(0) characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.