Search

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. [ABSTRACT FROM AUTHOR]

The nonheme iron dioxygenase capreomycin C (CmnC) hydroxylates a free L-arginine amino acid regio- and stereospe-cifically at the C3-position as part of the capreomycin antibiotics biosynthesis. Little is known on its structure, catalytic cycle and substrate specificity and, therefore, a comprehensive computational study was performed. A large QM cluster model of CmnC was created of 297 atoms and the mechanisms for C3-H, C4-H and C5-H hydroxylation and C3-C4 desaturation were investigated. All low-energy pathways correspond to radical reaction mechanisms with an initial hydrogen atom abstraction followed by OH rebound to form alcohol product complexes. The work is compared to alternative L-Arg hydroxylating nonheme iron dioxygenases and the differences in active site polarity are compared. We show that a tight hydrogen bonding network in the substrate binding pocket positions the substrate in an ideal orientation for C3-H activation, whereby the polar groups in the substrate binding pocket induce an electric field effect that guides the selectivity., (© 2024 Wiley‐VCH GmbH.)