Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase

• Published in: The FEBS journal 2024-04, Vol.291 (7), p.1404-1421
• Main Authors: Taylor, Aoife, Zhang, Shaowei, Johannissen, Linus O., Sakuma, Michiyo, Phillips, Robert S., Green, Anthony P., Hay, Sam, Heyes, Derren J., Scrutton, Nigel S.
• Format: Article
• Language: English
• Subjects: Binding; Biosynthesis; Catalysis; catalytic mechanism; Chlorophyll; chlorophyll biosynthesis; Electron microscopy; Enzymes; Glutamine; Oxidoreductase; Photocatalysis; Photochemistry; Protochlorophyllide; protochlorophyllide oxidoreductase; Protons; Reaction mechanisms; Structural models; structure; Substrates; Tyrosine
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.17025

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H‐transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme–substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water‐mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal ‘binding pose’ for the POR ternary enzyme–substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme. Photoenzymes harness light for catalysis but are rare in nature. Improving our understanding of how light energy facilitates enzyme catalysis is now becoming crucial for the design and engineering of non‐natural photo‐biocatalysts. Here, we examined different structural models of the photoenzyme protochlorophyllide oxidoreductase (POR) by experimental and computational approaches. A new structural model for the cyanobacterial POR ternary enzyme‐substrate complex is presented, together with a discussion of the implications for the potential mechanism of how this unique enzyme uses light energy to facilitate enzyme catalysis.