Pigment Binding in The Light-Dependent Protochlorophyllide Oxidoreductase

Abstract

AbstractThe Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) is a key enzyme in chlorophyll biosynthesis and its photocatalytic mechanism has long intrigued researchers. However, the lack of structural data for the active complex has impeded understanding of its reaction mechanism. A recent high-resolution structure of enzyme in the active conformation has established a robust foundation for validating hypotheses concerning pigment binding, residue involvement, and consequently, the reaction mechanism. Surprisingly, this new structure challenges previously proposed mechanisms, especially concerning the orientation of the bound protochlorophyllide (Pchlide) pigment. In this study, we employ molecular dynamics and hybrid quantum-mechanics/molecular-mechanics (QM/MM) simulations along with site-directed mutagenesis to compare two Pchlide binding modes: one aligned with previous proposals (mode A), and the other consistent with the recent experimental data (mode B). Binding energy calculations reveal thermodynamic instability of binding mode A due to nonspecific interactions, while mode B exhibits distinct stabilizing interactions yielding favorable binding. QM/MM-based local energy decomposition analysis unravels a complex interaction network that reinforces pigment stabilization in this conformation. Notably, interactions involving Tyr177, His319, and the carboxyl group at C131influence the pigment’s excited state energy and potentially contributing to the substrate specificity of the enzyme. Our results uniformly favor binding mode B as represented in the new cryo-EM structure, over the previously assumed mode A. These findings challenge established interpretations and underscore the need for a comprehensive re-evaluation of the reaction mechanism of LPOR that correctly considers pigment interactions and substrate orientation in the binding pocket.Significance StatementA crucial step in the biosynthesis of the all-important photosynthetic pigment chlorophyll is the reduction of a double C=C bond in its precursor protochlorophyllide (PChlide). This is catalyzed by the Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) via an extremely rare example of a biological photocatalytic reaction. Understanding of the LPOR mechanism has been hindered by limited insight into the structure of its active complex. A recent high-resolution LPOR cryo-EM structure substantiates pigment binding, residue interactions, and the reaction mechanism, but contrasts markedly with all previous assumptions regarding the binding mode of the substrate PChlide. Using molecular dynamics simulations, quantum-mechanics/molecular-mechanics calculations, and mutagenesis, we compare and evaluate the two possible Pchlide binding modes, the one assumed previously (mode A) and the one supported by recent data (mode B). Our findings conclusively favor mode B, challenging prior assumptions and pointing toward novel mechanistic possibilities for this unique photocatalytic reaction.