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

Abstract

The photoenzyme protochlorophyliide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide (Pchlide) to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight about the light-driven reaction chemistry, a structural rationale for POR photocatalysis has proved more 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 Pchlide substrate and the architecture of the POR active site that have significant implications for the catalytic mechanism of Pchlide reduction. Here, we have used a combination of computational and experimental approaches to investigate the compatibility of each of these structural models with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR-Pchlide-NADPH ternary complex based on these findings. Through detailed site-directed mutagenesis studies we show that a strictly conserved Tyr residue, which has previously been proposed to act as the proton donor in POR photocatalysis, is not likely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site Cys residue 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 Gln residue is found to be 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.

Fluorination of a conserved tyrosine in POR offers new clues for proton transfer

“…In this issue of The FEBS Journal , Taylor and colleagues inspected the framework for understanding POR's catalytic mechanism using fluorinated tyrosine (FY), a tool least perturbing Tyr's binding ability but substantially modifying its protonation state [13]. The substitution of hydrogen by fluorine, the element with the smallest size of all non‐hydrogen atoms and the most electronegativity, in a phenolic ring confers the electron‐withdrawing ability, and thus the p K a value of the FY side chain hydroxyl group is lowered from ~ 10 (for the wild‐type Tyr) to ~ 8 for 3‐FY and to ~ 6 for 2,3,5‐FY, denoting that the proton is easier to lose.…”

“…Oxygen, nitrogen, and sulfur atoms are in red, blue, and yellow, respectively. The right panel depicts the current model [13]. The phenolic C3 fluorine is shown in cyan and circled.…”