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

Silva PJ - PeerJ (2014)

Bottom Line: The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe-4S], yielding a negatively-charged intermediate.The computed reaction barriers suggest that Fe-S cluster re-reduction should be the rate-limiting stage of the process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: REQUIMTE, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa , Rua Carlos da Maia, Porto , Portugal.

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.

No MeSH data available.


Influence of the Fe–S cluster reduction state on the protonation of neighboring amino acids.Arrangement of Asp147, His53 and His13B around the Fe–S cluster in the substrate-bound protein and protonation probabilities of these amino acids at different pH/electric potential.
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fig-6: Influence of the Fe–S cluster reduction state on the protonation of neighboring amino acids.Arrangement of Asp147, His53 and His13B around the Fe–S cluster in the substrate-bound protein and protonation probabilities of these amino acids at different pH/electric potential.

Mentions: Several important characteristics of the reaction mechanism cannot be derived from truncated active site models due to the lack of the protein-induced electrostatic field, which depends on the overall charge distribution in the protein (Stephens, Jollie & Warshel, 1996; Kamerlin & Warshel, 2010; Ribeiro, 2013). In the enzyme studied in this report, such characteristics include the likelihood of occurrence of the active protonated states of Asp274 and propionic side chain at physiological pH and the susceptibility of the Fe–S cluster redox potential to the solution pH. Continuum electrostatics computations on the protochlorophyllide oxidoreductase structure bearing each of the quantum-chemically-optimized intermediate allowed us to quantify these effects (see Computational Methods for details). The inclusion of the protein barely affects the way the redox potentials of the Fe–S cluster vary as the intermediate gains electrons/protons (Table 3), but has an important effect on the sensitivity of the Fe–S cluster redox state towards changes in pH: in all instances the predicted change in redox potential per pH unit corresponds to the uptake of 0.8 protons by the Fe–S surroundings upon the one-electron reduction of the cluster. Analysis of the correlations matrixes clearly shows that the reduction of the Fe–S clusters increases the probability of finding the neighboring His53A and His13B in their protonated states. At lower pH, Asp147A also tends to become protonated as the cluster is reduced (Fig. 6).


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

Silva PJ - PeerJ (2014)

Influence of the Fe–S cluster reduction state on the protonation of neighboring amino acids.Arrangement of Asp147, His53 and His13B around the Fe–S cluster in the substrate-bound protein and protonation probabilities of these amino acids at different pH/electric potential.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4157233&req=5

fig-6: Influence of the Fe–S cluster reduction state on the protonation of neighboring amino acids.Arrangement of Asp147, His53 and His13B around the Fe–S cluster in the substrate-bound protein and protonation probabilities of these amino acids at different pH/electric potential.
Mentions: Several important characteristics of the reaction mechanism cannot be derived from truncated active site models due to the lack of the protein-induced electrostatic field, which depends on the overall charge distribution in the protein (Stephens, Jollie & Warshel, 1996; Kamerlin & Warshel, 2010; Ribeiro, 2013). In the enzyme studied in this report, such characteristics include the likelihood of occurrence of the active protonated states of Asp274 and propionic side chain at physiological pH and the susceptibility of the Fe–S cluster redox potential to the solution pH. Continuum electrostatics computations on the protochlorophyllide oxidoreductase structure bearing each of the quantum-chemically-optimized intermediate allowed us to quantify these effects (see Computational Methods for details). The inclusion of the protein barely affects the way the redox potentials of the Fe–S cluster vary as the intermediate gains electrons/protons (Table 3), but has an important effect on the sensitivity of the Fe–S cluster redox state towards changes in pH: in all instances the predicted change in redox potential per pH unit corresponds to the uptake of 0.8 protons by the Fe–S surroundings upon the one-electron reduction of the cluster. Analysis of the correlations matrixes clearly shows that the reduction of the Fe–S clusters increases the probability of finding the neighboring His53A and His13B in their protonated states. At lower pH, Asp147A also tends to become protonated as the cluster is reduced (Fig. 6).

Bottom Line: The reaction mechanism begins with single-electron reduction of the substrate by the (Cys)3Asp-ligated [4Fe-4S], yielding a negatively-charged intermediate.The computed reaction barriers suggest that Fe-S cluster re-reduction should be the rate-limiting stage of the process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: REQUIMTE, Faculdade de Ciências da Saúde, Universidade Fernando Pessoa , Rua Carlos da Maia, Porto , Portugal.

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.

No MeSH data available.