Limits...
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.


Overall reaction mechanism.Comparison of the overall reaction mechanisms of light-dependent (central pathway) and dark-operative (bottom pathway) protochlorophyllide oxidoreductases. The rings have been labeled according to the IUPAC nomenclature (Moss, 1987).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4157233&req=5

fig-1: Overall reaction mechanism.Comparison of the overall reaction mechanisms of light-dependent (central pathway) and dark-operative (bottom pathway) protochlorophyllide oxidoreductases. The rings have been labeled according to the IUPAC nomenclature (Moss, 1987).

Mentions: The reaction mechanism of the light-dependent protochlorophyllide oxidoreductase has been extensively studied through experimental (Heyes & Hunter, 2004; Heyes et al., 2009; Heyes et al., 2011; Sytina et al., 2012) and computational (Heyes et al., 2009; Silva & Ramos, 2011) methods. In contrast, relatively little is known about the precise sequence of events taking place in the dark-operative protochlorophyllide oxidoreductase (dPCHOR). The enzyme contains two components: a homodimeric L-protein which performs ATP-dependent electron transfer reminiscent of that observed in nitrogenase Fe protein (Fujita & Bauer, 2000; Sarma et al., 2008), and a heterotetrameric component bearing the active site and a (Cys)3Asp-ligated [4Fe–4S] cluster which accepts electrons from the L-protein and functions as the electron donor to the protochlorophyllide substrate (Muraki et al., 2010; Bröcker et al., 2010). The peculiar ligation of the electron-transferring [4Fe–4S] cluster has been shown by site-directed mutagenesis to be crucial to the enzyme activity (Muraki et al., 2010), probably due to the lowering of its reduction potential below that of other [4Fe–4S] clusters (Kondo et al., 2011; Takano et al., 2011). The crystallographic structure of dPCHOR (Muraki et al., 2010; Bröcker et al., 2010) shows that the substrate binding site, while mostly lined by hydrophobic residues, contains a single conserved aspartate residue (Asp274) which is thought to be a proton-donor for the reaction. Two protons and two electrons are required (Fig. 1), which necessarily entails two separate reduction events (as the [4Fe–4S] cluster is a one-electron donor) and the presence of a second proton-donor. Asp274 is unlikely to act as the donor of the second proton, as it cannot be reprotonated due to the absence of pathways linking it to the solvent. The propionic acid side-chain present on the substrate C17 was therefore proposed as the second proton donor (Muraki et al., 2010). The intricacies of proton and electron transfer from dPChOR to its protochlorophyllide substrate have, however, remained unaddressed by experimental methods. In this report, we describe this reaction mechanism with the help of density-functional theory methods. The results allow the description of the sequence of the reduction/protonation events and also identify the factors governing the stereochemical outcome of this enzyme-catalyzed reaction. Comparisons of the energetic features of the intermediates with those of common biochemical redox intermediates suggest a simple explanation for the differences observed in the dark-catalyzed and light-dependent enzyme reactions.


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)

Overall reaction mechanism.Comparison of the overall reaction mechanisms of light-dependent (central pathway) and dark-operative (bottom pathway) protochlorophyllide oxidoreductases. The rings have been labeled according to the IUPAC nomenclature (Moss, 1987).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-1: Overall reaction mechanism.Comparison of the overall reaction mechanisms of light-dependent (central pathway) and dark-operative (bottom pathway) protochlorophyllide oxidoreductases. The rings have been labeled according to the IUPAC nomenclature (Moss, 1987).
Mentions: The reaction mechanism of the light-dependent protochlorophyllide oxidoreductase has been extensively studied through experimental (Heyes & Hunter, 2004; Heyes et al., 2009; Heyes et al., 2011; Sytina et al., 2012) and computational (Heyes et al., 2009; Silva & Ramos, 2011) methods. In contrast, relatively little is known about the precise sequence of events taking place in the dark-operative protochlorophyllide oxidoreductase (dPCHOR). The enzyme contains two components: a homodimeric L-protein which performs ATP-dependent electron transfer reminiscent of that observed in nitrogenase Fe protein (Fujita & Bauer, 2000; Sarma et al., 2008), and a heterotetrameric component bearing the active site and a (Cys)3Asp-ligated [4Fe–4S] cluster which accepts electrons from the L-protein and functions as the electron donor to the protochlorophyllide substrate (Muraki et al., 2010; Bröcker et al., 2010). The peculiar ligation of the electron-transferring [4Fe–4S] cluster has been shown by site-directed mutagenesis to be crucial to the enzyme activity (Muraki et al., 2010), probably due to the lowering of its reduction potential below that of other [4Fe–4S] clusters (Kondo et al., 2011; Takano et al., 2011). The crystallographic structure of dPCHOR (Muraki et al., 2010; Bröcker et al., 2010) shows that the substrate binding site, while mostly lined by hydrophobic residues, contains a single conserved aspartate residue (Asp274) which is thought to be a proton-donor for the reaction. Two protons and two electrons are required (Fig. 1), which necessarily entails two separate reduction events (as the [4Fe–4S] cluster is a one-electron donor) and the presence of a second proton-donor. Asp274 is unlikely to act as the donor of the second proton, as it cannot be reprotonated due to the absence of pathways linking it to the solvent. The propionic acid side-chain present on the substrate C17 was therefore proposed as the second proton donor (Muraki et al., 2010). The intricacies of proton and electron transfer from dPChOR to its protochlorophyllide substrate have, however, remained unaddressed by experimental methods. In this report, we describe this reaction mechanism with the help of density-functional theory methods. The results allow the description of the sequence of the reduction/protonation events and also identify the factors governing the stereochemical outcome of this enzyme-catalyzed reaction. Comparisons of the energetic features of the intermediates with those of common biochemical redox intermediates suggest a simple explanation for the differences observed in the dark-catalyzed and light-dependent enzyme reactions.

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.