Flavins are employed as redox cofactors and chromophores in a plethora of flavoenzymes. Their versatility is an outcome of intrinsic molecular properties of the isoalloxazine ring modulated by the ...protein scaffold and surrounding solvent. Thus, an investigation of isolated flavins with high‐level electronic‐structure methods and with error assessment of the calculated properties will contribute to building better models of flavin reactivity. Here, we benchmarked ground‐state properties such as electron affinity, gas‐phase basicity, dipole moment, torsion energy, and tautomer stability for lumiflavins in all biologically relevant oxidation and charge states. Overall, multiconfigurational effects are small and chemical accuracy is achieved by coupled‐cluster treatments of energetic properties. Augmented basis sets and extrapolations to the complete basis‐set limit are necessary for consistent agreement with experimental energetics. Among DFT functionals tested, M06‐2X shows the best performance for most properties, except gas‐phase basicity, in which M06 and CAM‐B3LYP perform better. Moreover, dipole moments of radical flavins show large deviations for all functionals studied. Tautomers with noncanonical protonation states are significantly populated at normal temperatures, adding to the complexity of modeling flavins. These results will guide future computational studies of flavoproteins and flavin chemistry by indicating the limitations of electronic‐structure methodologies and the contributions of multiple tautomeric states.
Flavins are essential biological cofactors. Here, we benchmark high‐level quantum chemical methods to model lumiflavin in all biologically relevant oxidation and charge states, comparing structural, energetic, electrical, and chemical properties. We found limitations of some electronic‐structure methods, as well as alternative protonation states that may challenge the canonical assignment of tautomeric equilibria in flavins.
Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q
) and drive four protons across the inner mitochondrial membrane, powering ...oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q
reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q
. Using cryo-EM, we reveal a Q
molecule occupying the full length of the Q-binding site in the 'active' (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q
binding pose. By comparing ligand-bound and ligand-free forms of the 'deactive' resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.
Metalloproteins play indispensable roles in biology owing to the versatile chemical reactivity of metal centres. However, studying their reactivity in many metalloproteins is challenging, as protein ...three-dimensional structure encloses labile metal centres, thus limiting their access to reactants and impeding direct measurements. Here we demonstrate the use of single-molecule atomic force microscopy to induce partial unfolding to expose metal centres in metalloproteins to aqueous solution, thus allowing for studying their chemical reactivity in aqueous solution for the first time. As a proof-of-principle, we demonstrate two chemical reactions for the FeS4 centre in rubredoxin: electrophilic protonation and nucleophilic ligand substitution. Our results show that protonation and ligand substitution result in mechanical destabilization of the FeS4 centre. Quantum chemical calculations corroborated experimental results and revealed detailed reaction mechanisms. We anticipate that this novel approach will provide insights into chemical reactivity of metal centres in metalloproteins under biologically more relevant conditions.
The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, ...which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.
Spin is the thing: Iron–sulfur proteins of the rubredoxin family only have one Fe center coordinated by four cysteine residues (see picture, Fe orange). A multiscale modeling approach is used to see ...if FeS bond dissociation in these iron–sulfur clusters occurs by heterolytic fission or homolytic cleavage. As Fe complexes can have near‐degenerate levels with different total spin, their spin states and spin crossovers must be characterized during the reaction.
Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool ...for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the β‐glucosidase Sfβgly (β‐d‐glucoside glucohydrolase; EC 3.2.1.21). These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfβgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfβgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfβgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins.
Mutations directed to seven hubs of the protein structure network of the β‐glucosidase Sfβgly reduced its thermostability. Moreover, mutations directed to the vicinity of a hub residue also caused significant decreases in the Sfβgly thermostability. On the contrary mutations directed to non‐hub residues had no effect. These results show that protein structure networks are robust, whereas attacks on central nodes cause network failure.
Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane ...is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7(XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.
Modeling the Hydrolysis of Iron-Sulfur Clusters Teixeira, Murilo H; Curtolo, Felipe; Camilo, Sofia R G ...
Journal of chemical information and modeling,
02/2020, Letnik:
60, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Iron-sulfur (FeS) clusters are essential metal cofactors involved in a wide variety of biological functions. Their catalytic efficiency, biosynthesis, and regulation depend on FeS stability in ...aqueous solution. Here, molecular modeling is used to investigate the hydrolysis of an oxidized (ferric) mononuclear FeS cluster by bare dissociation and water substitution mechanisms in neutral and acidic solution. First, approximate electronic structure descriptions of FeS reactions by density functional theory are validated against high-level wave function CCSD(T) calculations. Solvation contributions are included by an all-atom model with hybrid quantum chemical/molecular mechanical (QM/MM) potentials and enhanced sampling molecular dynamics simulations. The free energy profile obtained for FeS cluster hydrolysis indicates that the hybrid functional M06 together with an implicit solvent correction capture the most important aspects of FeS cluster reactivity in aqueous solution. Then, 20 reaction channels leading to two consecutive Fe-S bond ruptures were explored with this calibrated model. For all protonation states, nucleophilic substitution with concerted bond breaking and forming to iron is the preferred mechanism, both kinetic and thermodynamically. In neutral solution, proton transfer from water to the sulfur leaving group is also concerted. Dissociative reactions show higher barriers and will not be relevant for FeS reactivity when exposed to solvent. These hydrolysis mechanisms may help to explain the stability and catalytic mechanisms of FeS clusters of multiple sizes and proteins.
Spin is the thing: Iron-sulfur proteins of the rubredoxin family only have one Fe center coordinated by four cysteine residues (see picture, Feorange). A multiscale modeling approach is used to see ...if FeS bond dissociation in these iron-sulfur clusters occurs by heterolytic fission or homolytic cleavage. As Fe complexes can have near-degenerate levels with different total spin, their spin states and spin crossovers must be characterized during the reaction.
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