An Alternative Proposal for the Reaction Mechanism of Light-Dependent Protochlorophyllide Oxidoreductase

Abstract

Light-dependent protochlorophyllide oxidoreductase is one of the few known enzymes that require a quantum of light to start their catalytic cycle. Upon excitation, it uses NADPH to reduce the C 17 −C 18 in its substrate (protochlorophyllide) through a complex mechanism that has heretofore eluded precise determination. Isotopic labeling experiments have shown that the hydride-transfer step is very fast, with a small barrier close to 9 kcal mol −1 , and is followed by a proton-transfer step, which has been postulated to be the protonation of the product by the strictly conserved Tyr189 residue. Since the structure of the enzyme− substrate complex has not yet been experimentally determined, we first used modeling techniques to discover the actual substrate binding mode. Two possible binding modes were found, both yielding stable binding (as ascertained through molecular dynamics simulations) but only one of which placed the critical C17C18 bond consistently close to the NADPH pro-S hydrogen and to Tyr189. This binding pose was then used as a starting point for the testing of previous mechanistic proposals using time-dependent density functional theory. The quantum-chemical computations clearly showed that such mechanisms have prohibitively high activation energies. Instead, these computations showed the feasibility of an alternative mechanism initiated by excited-state electron transfer from the key Tyr189 to the substrate. This mechanism appears to agree with the extant experimental data and reinterprets the final protonation step as a proton transfer to the active site itself rather than to the product, aiming at regenerating it for another round of catalysis.

How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis

“…An alternative mechanism for POR was proposed during the reviewing process for this manuscript, which involves eT from Tyr193 to the photoactivated PChlide . This mechanism was calculated to have a lower barrier (Δ G ‡ = 46.4 kJ mol –1 ), but a redox active Tyr193 is not consistent with mutagenesis experiments, which have shown that while Tyr193 mutants show decreased activity at physiological temperatures, there is no significant difference in the rate of HYT in the Y193F variant at cryogenic temperatures compared to that in the wild type .…”

“…Such a conformational change is likely required prior to protonation, yet it is not clear at what point during the reaction this occurs. Note, however, that protonation might involve bridging water molecule(s), and other proton donors have also been proposed based on alternative PChlide binding poses, , although Tyr193 is the most likely proton donor in our published model . All calculations were performed in Gaussian 16 revision A03 .…”

Steered Molecular Dynamics Simulations Study on FABP4 Inhibitors

“…The run using umbrella sampling (US) methodology on molecule 6 was carried out employing the YASARA macro written by Silva ( , accessed on 28 February 2023) [ 58 ].…”

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

“…Cys309 is fully conserved throughout all POR enzymes and interestingly, when the equivalent residue in T. elongatus POR, Cys226, was previously mutated to a Ser residue it appeared to influence the hydride and proton transfer chemistry [23]. To complicate matters further, a recent computational study suggested yet another alternative mechanism [11]. In this scenario, Pchlide photochemistry leads to electron transfer from the conserved Tyr residue to the C18 position of Pchlide, followed by a subsequent proton transfer, also from the Tyr residue, then electron and hydride transfer from NADPH [11].…”