Biology subjects

Arantes, G. M.

Publications and source records attributed to Arantes, G. M..

7 recordsLinked to original sources

bioRxiv2026

Conformational gating at histidine junctions coordinates proton translocation in respiratory complex I

Respiratory complex I, a central enzyme in cellular metabolism, converts the free energy of NADH oxidation into a transmembrane proton-motive force to drive ATP synthesis, but the molecular mechanisms by which it couples redox catalysis to vectorial proton translocation remain unresolved. Here, we present high-resolution cryo-EM structures of complex I from Bos taurus captured under conditions designed to change the protonation states of residues in the membrane domain. Our structures reveal conformational rearrangements at key pathway junctions that reconfigure proton-transfer connections. In ND5, helical rearrangements switch the connectivity of histidine-248 between proton-uptake and proton-output pathways. In ND4, rotameric changes of histidine-220 alternately enable proton uptake or lateral proton transfer along the membrane domain. Combined with molecular simulations, our structures define gating mechanisms that impose directionality on proton transfer reactions and provide a framework for proton-coupled energy transduction in complex I.

biochemistry↗

bioRxiv2024

Redox-Activated Proton Transfer through a Redundant Network in the Qo Site of Cytochrome bc1

Proton translocation catalyzed by cytochrome bc1 (respiratory complex III) during coenzyme-Q redox cycling is a critical bioenergetic process, yet its detailed molecular mechanism remains in-completely understood. Even the specific groups mediating proton transfer following coenzyme-Q oxidation have not been established. In this study, the energetics of proton transfer through multiple proton-conducting wires recently identified in the Qo site was investigated across various reactant redox states using hybrid QM/MM simulations and a specialized reaction coordinate. Key reactive groups and proton transfer mechanisms were characterized, confirming the propionate-A group of heme bL as a plausible proton acceptor. Upon quinol oxidation, a Grotthuss hopping mechanism is activated, facilitating proton transfer along three distinct pathways with comparable energetics. These pathways function redundantly, forming a robust proton-conducting network. A highly conserved tyrosine residue (Y147 in R. sphaeroides numbering) was found to be essential for complete proton transfer, whereas participation of H276 and D278 does not appear energetically feasible. Bioenergetic analyses exclude charged closed-shell species as likely intermediates and propose a reaction sequence for quinol oxidation proceeding as QH2 [->] QH* [->] Q0, either via coupled proton-electron transfers or stepwise mechanisms involving open-shell intermediates. These findings elucidate mechanistic details of the Q-cycle and improve our understanding of the catalytic reactions supporting coenzyme-Q redox cycling in respiratory complexes.

biophysics↗

bioRxiv2024

Flexibility and Hydration of the Qo Site Determine Multiple Pathways for Proton Transfer in Cytochrome bc1

The detailed catalytic activity of cytochrome bc1 (or respiratory complex III) and the molecular mechanism of the Q cycle remain elusive. At the Qo site, the cycle begins with oxidation of the coenzyme-Q substrate (quinol form) in a bifurcated two-electron transfer to the iron-sulfur (FeS) cluster and the heme bL center. The uptake of the two protons released during quinol oxidation is less understood, with one proton likely delivered to the histidine side chain attached to the FeS cluster. Here, we present extensive molecular dynamics simulations with enhanced sampling of side-chain torsions at the Qo site and analyze available sequences and structures of several bc1 homologues to probe the interactions of quinol with potential proton acceptors and identify viable pathways for proton transfer. Our findings reveal that side chains at the Qo site are highly flexible and can adopt multiple conformations. Consequently, the quinol head is also flexible, adopting three distinct binding modes. Two of these modes are proximal to the heme bL and represent reactive conformations capable of electron and proton transfer, while the third, more distal mode likely represents a pre-reactive state, consistent with recent cryo-EM structures of bc1 with bound coenzyme-Q. The Qo site is highly hydrated, with several water molecules bridging interactions between the quinol head and the conserved side chains Tyr147, Glu295, and Tyr297 in cytochrome b (numbering according to R. sphaeroides), facilitating proton transfer. A hydrogen bond network and at least five distinct proton wires are established and possibly transport protons via a Grotthuss mechanism. Asp287 and propionate-A of heme bL in cytochrome b are in direct contact with external water and are proposed as the final proton acceptors. The intervening water molecules in these proton wires exhibit low mobility, and some have been resolved in recent experimental structures. These results help to elucidate the intricate molecular mechanism of the Q-cycle and pave the way to a detailed understanding of chemical proton transport in several bioenergetic enzymes that catalyze coenzyme-Q redox reactions.

biophysics↗

bioRxiv2022

Cryo-EM structures reveal ubiquinone-10 binding to mitochondrial complex I and conformational transitions associated with Q-site occupancy

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, structures of Q10-bound states have remained elusive. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the active (ready-to-go) resting state (plus a matching substrate-free structure) and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 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.

biochemistry↗

bioRxiv2019

Combining free energy simulations and NMR chemical-shift perturbation to identify transient cation-π contacts in proteins

Flexible protein regions containing cationic and aromatic side-chains exposed to solvent may form transient cation-{pi} interactions with structural and functional roles. To evaluate their stability and identify important intramolecular cation-{pi} contacts, a combination of free energy profiles estimated from umbrella sampling with molecular dynamics simulations and chemical shift perturbations (CSP) obtained from NMR experiments is applied here to the complete catalytic domain of human phosphatase Cdc25B. This protein is a good model system for transient cation-{pi} interactions as it contains only one Trp residue (W550) in the disordered C-terminal segment and a total of 17 Arg residues, many exposed to solvent. Eight putative Arg-Trp pairs were simulated here. Only R482 and R544 show bound profiles corresponding to important transient cation-{pi} interactions, while the others have dissociative or almost flat profiles. These results are corroborated by CSP analysis of three Cdc25B point mutants (W550A, R482A and R544A) disrupting cation-{pi} contacts. The proposed validation of statistically representative molecular simulations by NMR spectroscopy could be applied to identify transient contacts of proteins in general but carefully, as NMR chemical shifts are sensitive to changes in both molecular contacts and conformational distributions.

biophysics↗

bioRxiv2019

Balanced internal hydration discriminates substrate binding to respiratory complex I

Molecular recognition of the amphiphilic electron carrier ubiquinone (Q) by respiratory complexes is a fundamental part of electron transfer chains in mitochondria and many bacteria. The primary respiratory complex I binds Q in a long and narrow protein chamber to catalyse its reduction. But, the binding mechanism and the role of chamber hydration in substrate selectivity and stability are unclear. Here, large-scale atomistic molecular dynamics simulations and estimated free energy profiles are used to characterize in detail the binding mechanism to complex I of Q with short and with long isoprenoid tails. A highly stable binding site with two different poses near the chamber exit and a secondary reactive site near the N2 iron-sulfur cluster are found which may lead to an alternative Q redox chemistry and help to explain complex I reactivity. The binding energetics depends mainly on polar interactions of the Q-head and on the counterbalanced hydration of Q-tail isoprenoid units and hydrophobic residues inside the protein chamber. Selectivity upon variation of tail length arises by shifting the hydration balance. This internal hydration mechanism may have implications for binding of amphiphilic molecules to cavities in other membrane proteins.

biophysics↗

bioRxiv2019

Effects of lipid composition on membrane distribution and permeability of natural quinones

Natural quinones are amphiphilic molecules that function as mobile charge carriers in biological energy transduction. Their distribution and permeation across membranes are important for binding to enzymatic complexes and for proton translocation. Here, we employ molecular dynamics simulations and free energy calculations with a carefully calibrated classical force-field to probe quinone distribution and permeation in a multicomponent bilayer trying to mimic the composition of membranes involved in bioenergetic processes. Ubiquinone, ubiquinol, plastoquinone and menaquinone molecules with short and long isoprenoid tails are simulated. We find that water penetration increases considerably in the less ordered and porous bilayer formed by di-linoleoyl (18:2) phospholipids, resulting in a lower free energy barrier for quinone permeation and faster transversal diffusion. In equilibrium, quinone and quinol heads localize preferentially near lipid glycerol groups, but do not perform specific contacts with lipid polar heads. Quinone distribution is not altered significantly by the quinone head, tail and lipid composition in comparison to a single-component bilayer. This study highlights the role of acyl chain unsaturation for molecular permeation and transversal diffusion across biological membranes.

biophysics↗