Activation of a Photoenzyme Results in Modified Structure and Dynamics

Abstract

The interplay between protein dynamics and catalysis remains a fundamental question in enzymology. Here, we investigate the ns-timescale dynamics and solution structures of a light-dependent NADPH: protochlorophyllide oxidoreductase (LPOR), a photoenzyme crucial for chlorophyll synthesis. Due to the lack of an LPOR structure, the global structural and dynamic consequences of LPOR/Pchlide/NADPH ternary complex formation remained elusive up to now. By employing quasielastic neutron scattering (QENS) we show that the formation of the ternary holoprotein complex as well as photoactivation lead to progressive rigidification of the protein. Molecular dynamics (MD) simulations, in good agreement with the experimental QENS results, suggests that the increased flexibility observed for the apoprotein stems from structural fluctuations of the NADPH and Pchlide substrate binding sites of the enzyme. In addition, we investigated structural properties of the apo and holoproteins using MD simulations, multi-wavelength analytical ultracentrifugation (MWA-AUC) and small angle X-ray scattering (SAXS) experiments to build a consensus model of the LPOR apoprotein and the substrate/cofactor/LPOR ternary complex. MWA-AUC and SAXS experiments independently demonstrate that the apoprotein is monomeric, while ternary complex formation induces dimerization. SAXS-guided modelling studies provide a full-length model of the apoprotein and suggest a tentative mode of dimerization for the LPOR ternary complex, supported by published cross-link constraints. Our study provides a first impression of the LPOR structural organization and the relevance of molecular dynamics for the function of that photoenzyme.