The four-electron reduction of dioxygen to water in multicopper oxidases takes place in a trinuclear copper cluster, which is linked to a mononuclear blue copper site, where the substrates are oxidized. Recently, several intermediates in the catalytic cycle have been spectroscopically characterized, and two possible structural models have been suggested for both the peroxy and native intermediates. In this study, these spectroscopic results are complemented by hybrid quantum and molecular mechanical (QM/MM) calculations, taking advantage of recently available crystal structures with a full complement of copper ions. Thereby, we obtain optimized molecular structures for all of the experimentally studied intermediates involved in the reductive cleavage of the O(2) molecule and energy profiles for individual reaction steps. This allows identification of the experimentally observed intermediates and further insight into the reaction mechanism that is probably relevant for the whole class of multicopper oxidases. We suggest that the peroxy intermediate contains an O(2)(2-) ion, in which one oxygen atom bridges the type 2 copper ion and one of the type 3 copper ions, whereas the other one coordinates to the other type 3 copper ion. One-electron reduction of this intermediate triggers the cleavage of the O-O bond, which involves the uptake of a proton. The product of this cleavage is the observed native intermediate, which we suggest to contain a O(2-) ion coordinated to all three of the copper ions in the center of the cluster.
Hydrogen atom abstraction (HAA) reactions are cornerstones of chemistry. Various (metallo)enzymes performing the HAA catalysis evolved in nature and inspired the rational development of multiple synthetic catalysts. Still, the factors determining their catalytic efficiency are not fully understood. Herein, we define the simple thermodynamic factor η by employing two thermodynamic cycles: one for an oxidant (catalyst), along with its reduced, protonated, and hydrogenated form; and one for the substrate, along with its oxidized, deprotonated, and dehydrogenated form. It is demonstrated that η reflects the propensity of the substrate and catalyst for (a)synchronicity in concerted H+/e− transfers. As such, it significantly contributes to the activation energies of the HAA reactions, in addition to a classical thermodynamic (Bell–Evans–Polanyi) effect. In an attempt to understand the physicochemical interpretation of η, we discovered an elegant link between η and reorganization energy λ from Marcus theory. We discovered computationally that for a homologous set of HAA reactions, λ reaches its maximum for the lowest |η|, which then corresponds to the most synchronous HAA mechanism. This immediately implies that among HAA processes with the same reaction free energy, ΔG0, the highest barrier (≡ΔG≠) is expected for the most synchronous proton-coupled electron (i.e., hydrogen) transfer. As proof of concept, redox and acidobasic properties of nonheme FeIVO complexes are correlated with activation free energies for HAA from C−H and O−H bonds. We believe that the reported findings may represent a powerful concept in designing new HAA catalysts.
The stereoselectivity of the reaction of furan (1) with maleic anhydride (2) and maleimide (3) was studied experimentally and theoretically. Although the two reactions are highly similar with regard to their preference for endo and exo steroisomers, notable differences were experimentally observed and explained on the basis of calculated reaction-free energies and transition-state barriers. The experimental values of rate constants (k(1+2endo) = (1.75 +/- 0.48) x 10(-5); mol(-1) l s(-1); k(1+2exo) = (3.10 +/- 0.55) x 10(-5); mol(-1) l s(-1); k(1+3endo) = (1.93 +/- 0.082) x 10(-5); mol(-1) l s(-1), k(1+3exo) = (1.38 +/- 0.055) x 10(-5); mol(-1) l s(-1) all at 300 K) and the observed reaction course clearly confirm that neither of these reactions are prototypical examples of Diels-Alder [4 + 2] cycloadditions, whose dominant preference is for endo isomers. However, only by comparing their energetics-calculated at the CCSD(T) level of theory-with the analogous reactions involving cyclopentadiene (8) as a diene can these observations be understood. The low thermodynamic stability of furan [4 + 2] adducts opens retro-Diels-Alder reaction channels and overrules the very small kinetic preference (calculated and measured here) of initial formation for endo stereoisomers. On a macroscopic scale "an irregular"-thermodynamically more stable-exo stereoisomer was consequently observed as a dominant species.
Cyclic dinucleotides are second messengers in the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2′–5′,3′–5′-cyclic dinucleotides (2′3′CDNs) with use of cyclic GMP–AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5′-triphosphate (NTP) analogues and use them to prepare 33 2′3′CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure–activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
The ability to charge huge biomolecules without breaking them apart has made matrix-assisted laser desorption/ionization (MALDI) mass spectrometry an indispensable tool for biomolecular analysis. Conventional, empirically selected matrices produce abundant matrix ion clusters in the low-mass region (<500 Da), hampering the application of MALDI-MS to metabolomics. An ionization mode of MAILD, a rational protocol for matrix selection based on Brønsted-Lowry acid-base theory and its application to metabolomics, biological screening/profiling/imaging, and clinical diagnostics is illustrated. Numerous metabolites, covering important metabolic pathways (Krebs' cycle, fatty acid and glucosinolate biosynthesis), were detected in extracts, biofluids, and/or in biological tissues (Arabidopsis thaliana, Drosophila melanogaster, Acyrthosiphon pisum, and human blood). This approach moves matrix selection from ''black art'' to rational design and sets a paradigm for small-molecule analysis via MALDI-MS. and electrospray ionization-mass spectrometry (3) (ESI-MS) have been at the forefront of bioanalytical research with farreaching applications in proteomics (4), genomics (5), biological imaging (6), and metabolomics (7). In MALDI-MS, biomolecules mixed with matrices (small, UV-absorbing compounds) and exposed to laser pulses form gas-phase ions that are typically measured in time-of-flight (TOF) mass analyzers. Although the highthroughput nature of MALDI-MS makes it an ideal tool for large-scale metabolomic studies, its application in the field has been rather limited. This is because all conventional matrices (8-10) produce a forest of interfering low-mass ions (Ͻ500 Da) obscuring the detection of metabolites in the range. Despite several approaches (11-13), challenging MALDI-MS-based metabolomic tasks such as direct biofluid analysis, on-tissue metabolite screening, and generation of snapshots of the metabolic machinery of biological systems remains an unmet challenge.These limitations call for matrices devoid of interfering ions (''ionless matrices''), yet still assisting an efficient ionization/ desorption of the analytes. An ideal solution would be to have a rational selection protocol for such matrices, whereby depending on the properties of the analytes of interest, appropriate matrices could be designed. Such a development would not only cross a long-lasting hurdle of empirical selection of MALDI matrices but would also provide a powerful, fast, and easy-to-use tool to the biological community to selectively probe into the metabolomes of living organisms.Here, we report on a first-ever rational selection protocol for matrix-assisted mass spectrometry matrices based on the classical Brønsted-Lowry acid-base theory (14) and density functional theory (DFT) quantum chemical calculations. The matrices developed herein are ionless, in other words, the matrices produce no interfering matrix-related ions, thus overcoming the problem of most conventional matrices and allowing the detection of small molecules (0-1,000 Da). Furth...
The catalytic cycle of multicopper oxidases (MCOs) involves intramolecular electron transfer (IET) from the Cu‐T1 copper ion, which is the primary site of the one‐electron oxidations of the substrate, to the trinuclear copper cluster (TNC), which is the site of the four‐electron reduction of dioxygen to water. In this study we report a detailed characterization of the kinetic and electrochemical properties of bilirubin oxidase (BOx) – a member of the MCO family. The experimental results strongly indicate that under certain conditions, e.g. in alkaline solutions, the IET can be the rate‐limiting step in the BOx catalytic cycle. The data also suggest that one of the catalytically relevant intermediates (most likely characterized by an intermediate oxidation state of the TNC) formed during the catalytic cycle of BOx has a redox potential close to 0.4 V, indicating an uphill IET process from the T1 copper site (0.7 V) to the Cu‐T23. These suggestions are supported by calculations of the IET rate, based on the experimentally observed Gibbs free energy change and theoretical estimates of reorganization energy obtained by combined quantum and molecular mechanical (QM/MM) calculations.
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.
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