With or without light: comparing the reaction mechanism of dark-operative protochlorophyllide oxidoreductase with the energetic requirements of the light-dependent protochlorophyllide oxidoreductase

Abstract

The addition of two electrons and two protons to the C17=C18 bond in protochlorophyllide is catalyzed by a light-dependent enzyme relying on NADPH as electron donor, and by a light-independent enzyme bearing a (Cys)3Asp-ligated [4Fe–4S] cluster which is reduced by cytoplasmic electron donors in an ATP-dependent manner and then functions as electron donor to protochlorophyllide. The precise sequence of events occurring at the C17=C18 bond has not, however, been determined experimentally in the dark-operating enzyme. In this paper, we present the computational investigation of the reaction mechanism of this enzyme at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level of theory. The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe–4S], yielding a negatively-charged intermediate. Depending on the rate of Fe–S cluster re-reduction, the reaction either proceeds through double protonation of the single-electron-reduced substrate, or by alternating proton/electron transfer. The computed reaction barriers suggest that Fe–S cluster re-reduction should be the rate-limiting stage of the process. Poisson–Boltzmann computations on the full enzyme–substrate complex, followed by Monte Carlo simulations of redox and protonation titrations revealed a hitherto unsuspected pH-dependence of the reaction potential of the Fe–S cluster. Furthermore, the computed distributions of protonation states of the His, Asp and Glu residues were used in conjuntion with single-point ONIOM computations to obtain, for the first time, the influence of all protonation states of an enzyme on the reaction it catalyzes. Despite exaggerating the ease of reduction of the substrate, these computations confirmed the broad features of the reaction mechanism obtained with the medium-sized models, and afforded valuable insights on the influence of the titratable amino acids on each reaction step. Additional comparisons of the energetic features of the reaction intermediates with those of common biochemical redox intermediates suggest a surprisingly simple explanation for the mechanistic differences between the dark-catalyzed and light-dependent enzyme reaction mechanisms.

Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase

“…Inter lies 2.4 kcal mol −1 higher in energy than TS1 and is able to reversibly rearrange back to the reactive complex essentially without a barrier. That TS’s relative energy is lower than Inter’s is a common artifact of the use of zero-point vibrational energy (ZPVE) correction and/or empirical corrections on flat potential energy surface313233. Interestingly, in our specific system, the stabilization effect on TS1 from ZPVE correction happens to be more significant than that of Inter.…”

Refining the reaction mechanism of O2towards its co-substrate in cofactor-free dioxygenases

“…As in previous works by our and other groups (Silva & Ramos, 2008; Silva & Ramos, 2009; Silva, 2014; Wijaya et al, 2016) reorganization energies for every molecule in both oxidation states were computed using the water-optimized reactant geometries for the product state (and vice-versa) and activation energies were then computed by building appropriate Marcus parabolas using these reorganization energies. The smaller size of these models allowed us to increase the size of the basis set in these computations to 6-311G(d,p), while keeping the diffuse functions on the oxygen atoms to allow a better description of the oxygen-based anionic species (henceforth this basis set will be referred to as BS2).…”

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

“…LPOR belongs to the short-chain dehydrogenase/reductase superfamily, whereas DPOR is, surprisingly, genetically and evolutionarily related to nitrogenase, a multi-subunit metalloenzyme capable of converting atmospheric nitrogen into ammonia. Despite relatively low identity at the amino acid level, the crystal structures of both nitrogenase − and DPOR , not only share overall structure similarities but also interact with related Fe–S proteins, which act as their electron donors upon ATP hydrolysis. , Although extensive research on LPOR and DPOR has been carried out at the molecular level over the past two decades, ,− the precise details of their mechanisms still remain insufficiently described. It is generally assumed that LPOR uses light absorbed by its substrate PChlide to prime its cofactor NADPH for hydride transfer from the pro-S face of its nicotinamide ring to the C 17 position of the PChlide molecule, followed by a proton transfer reaction from a conserved Tyr residue to the C 18 position of PChlide.…”