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- Chemiosmotic misunderstandingsPublication . Silva, Pedro J.Recent publications have questioned the appropriateness of the chemiosmotic theory, a key tenet of modern bioenergetics originally described by Mitchell and since widely improved upon and applied. In one of them, application of Gauss’ law to a model charge distribution in mitochondria was argued to refute the possibility of ATP generation through H+ movement in the absence of a counterion, whereas a different author advocated, for other reasons, the impossibility of chemiosmosis and proposed that a novel energy-generation scheme (referred to as “murburn”) relying on superoxide-catalyzed (or superoxide-promoted) ADP phosphorylation would operate instead. In this letter, those proposals are critically examined and found to be inconsistent with established experimental data and new theoretical calculations.
- Response to “molecular-level understanding of biological energy coupling and transduction: response to “chemiosmotic misunderstandings”Publication . Silva, Pedro J.The most recent contribution by Sunil Nath in these pages is, mostly, a repetition of his previous claims regarding failures of the chemiosmotic hypotheses, supplemented with some fresh misunderstandings of the points I had sought to clarify in my previous critique. Considerable portions rehash 50-60 years-old controversies, with no apparent understanding that the current chemiosmotic hypothesis, while birthed by Mitchell, differs from Mitchell's details in many respects. As such, Nath has devoted much time dealing with a few errors (or wrong hypotheses) by Mitchell (in a few places I would almost venture to say "typographical mistakes in typesetting") and presents the ensuing conclusions as "refutations" of the chemiosmotic paradigm, completely neglecting that such details (such as the precise H+/ATP or H+:O ratios) are completely irrelevant to the reality (or not) of an electron-transport chain that uses the free energy liberated by electron-transfer to remove H+ from a compartment, to which it returns through and ATP synthase which uses the energy in that spontaneous return to drive ATP synthesis. The thermodynamical mistakes and misunderstandings of the relevant literature present in Nath's new contribution are so numerous, though, that I feel forced to call the attention of the readers of "Biophysical Chemistry" to them.
- Computational development of rubromycin-based lead compounds for HIV-1 reverse transcriptase inhibitionPublication . Bernardo, Carlos E. P.; Silva, Pedro J.The binding of several rubromycin-based ligands to HIV1-reverse transcriptase was analyzed using molecular docking and molecular dynamics simulations. MM-PBSA analysis and examination of the trajectories allowed the identification of several promising compounds with predicted high affinity towards reverse transcriptase mutants which have proven resistant to current drugs. Important insights on the complex interplay of factors determining the ability of ligands to selectively target each mutant have been obtained.
- Computational improvement of small-molecule inhibitors of Bacillus anthracis protective antigen activation through isostere-based substitutionsPublication . Silva, Pedro J.; Silva, Sandra V.R.L.There has recently been interest in the development of small-molecule inhibitors of the oligomerization of Bacillus anthracis protective antigen for therapeutic use. Some of the proposed lead compounds have, however, unfavorable solubility in aqueous medium, which prevents their clinical use. In this computational work, we have designed several hundreds of derivatives with progressively higher hydro-solubility and tested their ability to dock the relevant binding cavity. The highest-ranking docking hits were then subjected to 125 nslong simulations to ascertain the stability of the binding modes. Several of the potential candidates performed quite disappointingly, but two molecules showed very stable binding modes throughout the complete simulations. Besides the identification of these two promising leads, these molecular dynamics simulations allowed the discovery of several insights that shall prove useful in the further improvement of these candidate towards higher potency and stability.
- An alternative proposal for the reaction mechanism of light-dependent protochlorophyllide oxidoreductasePublication . Silva, Pedro J.; Cheng, QiLight-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 C17−C18 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.
- An alternative proposal for the reaction mechanism of light-dependent protochlorophyllide oxidoreductasePublication . Silva, Pedro J.; Cheng, QiLight-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 C17–C18 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.
- With or without light: comparing the reaction mechanism of dark-operative protochlorophyllide oxidoreductase with the energetic requirements of the light-dependent protochlorophyllide oxidoreductasePublication . Silva, Pedro J.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.
- Computational development of inhibitors of plasmid-borne bacterial dihydrofolate reductasePublication . Silva, Pedro J.Resistance to trimethoprim and other antibiotics targeting dihydrofolate reductase may arise in bacteria harboring an atypical, plasmid-encoded, homotetrameric dihydrofolate reductase, called R67 DHFR. Although developing inhibitors to this enzyme may be expected to be promising drugs to fight trimethoprim-resistant strains, there is a paucity of reports describing the development of such molecules. In this manuscript, we describe the design of promising lead compounds to target R67 DHFR. Density-functional calculations were first used to identify the modifications of the pterin core that yielded derivatives likely to bind the enzyme and not susceptible to being acted upon by it. These unreactive molecules were then docked to the active site, and the stability of the docking poses of the best candidates was analyzed through triplicate molecular dynamics simulations, and compared to the binding stability of the enzyme–substrate complex. Molecule 32 ([6-(methoxymethyl)-4-oxo-3,7-dihydro-4H-pyrano[2,3-d]pyrimidin-2- yl]methyl-guanidinium) was shown by this methodology to afford extremely stable binding towards R67 DHFR and to prevent simultaneous binding to the substrate. Additional docking and molecular dynamics simulations further showed that this candidate also binds strongly to the canonical prokaryotic dihydrofolate reductase and to human DHFR, and is therefore likely to be useful to the development of chemotherapeutic agents and of dual-acting antibiotics that target the two types of bacterial dihydrofolate reductase.
- How to tune the absorption spectrum of chlorophylls to enable better use of the available solar spectrumPublication . Silva, Pedro J.; Osswald-Claro, Maria; Castro Mendonça, RosárioPhoton capture by chlorophylls and other chromophores in light-harvesting complexes and photosystems is the driving force behind the light reactions of photosynthesis. Excitation of photosystem II allows it to receive electrons from the water-oxidizing oxygen-evolution complex and to transfer them to an electron-transport chain that generates a transmembrane electrochemical gradient and ultimately reduces plastocyanin, which donates its electron to photosystem I. Subsequently, excitation of photosystem I leads to electron transfer to a ferredoxin which can either reduce plastocyanin again (in so-called “cyclical electron-flow”) and release energy for the maintenance of the electrochemical gradient, or reduce NADP+ to NADPH. Although photons in the far-red (700–750 nm) portion of the solar spectrum carry enough energy to enable the functioning of the photosynthetic electron-transfer chain, most extant photosystems cannot usually take advantage of them due to only absorbing light with shorter wavelengths. In this work, we used computational methods to characterize the spectral and redox properties of 49 chlorophyll derivatives, with the aim of finding suitable candidates for incorporation into synthetic organisms with increased ability to use far-red photons. The data offer a simple and elegant explanation for the evolutionary selection of chlorophylls a, b, c, and d among all easily-synthesized singly-substituted chlorophylls, and identified one novel candidate (2,12-diformyl chlorophyll a) with an absorption peak shifted 79 nm into the far-red (relative to chlorophyll a) with redox characteristics fully suitable to its possible incorporation into photosystem I (though not photosystem II). chlorophyll d is shown by our data to be the most suitable candidate for incorporation into far-red utilizing photosystem II, and several candidates were found with red-shifted Soret bands that allow the capture of larger amounts of blue and green light by light harvesting complexes.
- A tale of two acids: when arginine is a more appropriate acid than H3O+Publication . Silva, Pedro J.; Schulz, Claudia; Jahn, Dieter; Jahn, Martina; Ramos, Maria JoãoUroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen-III to yield coproporphyrinogen-III. We have previously found that the rate-limiting step is substrate protonation, rather than decarboxylation itself, and that this protonation can be effected by a nearby arginine residue (Arg37). In this report, we have studied the reasons for the unusual choice of arginine as a general acid catalyst. Our density functional calculations show that, although substrate protonation by H3O+ is both exergonic and very fast, in the presence of a protonated Arg37 substrate decarboxylation becomes rate-limiting and the substrate spontaneously breaks upon protonation. These results suggest that the active site must be shielded from solvent protons, and that therefore H3O+ should be excluded from a role in both protonations present in this mechanism. A second Arg residue (Arg41) is uniquely positioned to act as donor of the second proton, with an activation barrier below 2 kcal mol-1. Additional site-directed mutagenesis experiments confirmed that no coproporphyrinogen is formed in the absence of any of these these Arg residues. This counter-intuitive use of two basic residues as general acids in two different proton donation steps by uroporphyrinogen decarboxylase may have arisen as an elegant solution to the problem of simultaneously binding the very negative uroporphyrinogen (which requires a positively charged active site), and selectively protonating it while preventing excessive carboxylate stabilization by positive charges.
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