Biology subjects

Kaila, V. R. I.

Publications and source records attributed to Kaila, V. R. I..

8 recordsLinked to original sources

bioRxiv2024

Protein-Induced Membrane Strain Drives Supercomplex Formation

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III2 supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leading to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and strain effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions. Significance StatementThe membrane-bound proteins of respiratory chains power oxidative phosphorylation (OXPHOS) and drive the synthesis of ATP. However, recent biochemical and structural data show that the OXPHOS proteins operate as higher-order supercomplex assemblies for reasons that remain elusive and much debated. Here we show that the mammalian respiratory supercomplexes reduce the molecular strain of inner mitochondrial membranes and enhance the allosteric crosstalk by altering the protein dynamics with important biochemical and physiological implications.

biophysics↗

bioRxiv2024

Modified Chlorophyll Pigment at ChlD1 Tunes Photosystem II Beyond the Red-Light Limit

Photosystem II (PSII) is powered by the light-capturing properties of chlorophyll a pigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls for long wavelength pigments in specific locations, including one in the reaction center (RC). However, the exact location and the nature of this long wavelength pigment still remain uncertain. Here we have addressed the color-tuning mechanism of the farred light PSII (FRL-PSII) by excited state calculations at both the ab initio correlated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophyll d leads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1 site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.

biophysics↗

bioRxiv2024

Mechanism of Proton Release during Water Oxidation in Photosystem II

Photosystem II (PSII) catalyzes the light-driven water oxidation that releases dioxygen into our atmosphere and provides the electrons needed for the synthesis of biomass. The catalysis occurs in the oxygen-evolving oxo-manganese-calcium (Mn4O5Ca) cluster that drives the stepwise oxidation and deprotonation of substrate water molecules leading to the O2 formation. However, despite recent advances, the mechanism of these reactions remains unclear and much debated. Here we show that the light-driven Tyr161D1 oxidation adjacent to the Mn4O5Ca cluster, significantly decreases the barrier for proton transfer from the putative substrate water molecule (W3/Wx) to Glu310D2, which is accessible to the luminal bulk. By combining hybrid quantum/classical (QM/MM) free energy calculations with atomistic molecular dynamics (MD) simulations, we probe the energetics of the proton transfer along the Cl1 pathway. We demonstrate that the proton transfer occurs via water molecules and a cluster of conserved carboxylates, driven by redox-triggered electric fields directed along the pathway. Glu65D1 establishes a local molecular gate that controls the proton transfer to the luminal bulk, whilst Glu312D2 acts as a local proton storage site. The identified gating region could be important in preventing back-flow of protons to the Mn4O5Ca cluster. The structural changes, derived here based on the dark-state PSII structure, strongly support recent time-resolved XFEL data of the S3[->]S4 transition (Nature 617, 2023), and reveal the mechanistic basis underlying deprotonation of the substrate water molecules. Our combined findings provide insight into the water oxidation mechanism of PSII and show how the interplay between redox-triggered electric fields, ion-pairs, and hydration effects control proton transport reactions. Significance StatementPhotosystem II is natures water splitting enzyme that produces the oxygen in the atmosphere and drives the synthesis of biomass. The water splitting reaction releases protons to the luminal bulk contributing to the protonmotive force that drives the synthesis of ATP. Key mechanistic principles of the light-driven water splitting reaction remain debated, amongst them the catalytically important deprotonation steps. Here we show how the oxygen-evolving oxo-manganese-calcium cluster transports protons via conserved carboxylates and water molecules in proton arrays that lead to the luminal bulk. We identify a local proton storage site and molecular gates that prevent wasteful back reactions by undergoing conformational changes, and we show how electric field effects control the protonation dynamics in Photosystem II.

biophysics↗

bioRxiv2024

Molecular principles of redox-coupled sodium pumping of the ancient Rnf machinery

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD+ and pumps sodium ions (Na+) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na+-pumping NADH-quinone oxidoreductase (Nqr)1. By running in reverse, Rnf reduces ferredoxin with NADH as reductant at the expense of the transmembrane electrochemical ion gradient and provides low potential electrons for nitrogenases as well as CO2 reductases. Yet, the molecular principles that couple the long-range electron transfer to the Na+ translocation across the membrane remain elusive. Here we resolve key functional states along the electron transfer pathway using redox-controlled cryo-electron microscopy (cryo-EM) that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na+ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster in the vestibule between the RnfA/E subunits electrostatically attracts the sodium ions, and in turn, triggers an inward/outward transition with alternating membrane access driving the Na+ pump and the reduction of NAD+. Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.

biochemistry↗

bioRxiv2024

QM/MM Free Energy Calculations of Long-Range Biological Protonation Dynamics by Adaptive and Focused Sampling

Water-mediated proton transfer reactions are central for catalytic processes in a wide range of biochemical systems, ranging from biological energy conversion to chemical transformations in the metabolism. Yet, the accurate computational treatment of such complex bio-chemical reactions is highly challenging and requires the application of multiscale methods, in particular hybrid quantum/classical (QM/MM) approaches combined with free energy simulations. Here we combine the unique exploration power of new advanced sampling methods with density functional theory (DFT)-based QM/MM free energy methods for multiscale simulations of long-range protonation dynamics in biological systems. In this regard, we show that combining multiple walkers/well-tempered metadynamics with an extended-system adaptive biasing force method (MWE), provides a powerful approach for exploration of water-mediated proton transfer reactions in complex biochemical systems. We compare and combine the MWE method also with QM/MM-umbrella sampling and explore the sampling of the free energy landscape with both geometric (linear combination of proton transfer distances) and physical (center of excess charge) reaction coordinates, and show how these affect the convergence of the potential of mean force (PMF) and the activation free energy. We find that the QM/MM-MWE method can efficiently explore both direct and water-mediated proton transfer pathways together with forward and reverse hole transfer mechanisms in the highly complex proton channel of respiratory Complex I, while the QM/MM-US approach shows a systematic convergence of selected long-range proton transfer pathways. In this regard, we show that the PMF along multiple proton transfer pathways is recovered by combining the strengths of both approaches in a QM/MM-MWE/focused US (FUS) scheme, and revealing new mechanistic insight into the proton transfer principles of Complex I. Our findings provide a promising basis for the quantitative multi-scale simulations of long-range proton transfer reactions in biological systems.

biophysics↗

bioRxiv2024

Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+-exchange across the membrane. Despite recent structural advances (1-4), the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here we probe how the redox chemistry drives the proton reduction to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L towards the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis, and how these effects are employed by the [NiFe] active site of Mbh to drive the PCET reactions and ion transport. Significance statementHydrogen (H2) serves as a crucial solar fuel in renewable energy systems that can be efficiently produced by microbial hydrogenases. Here we probe the elusive mechanistic principles underlying the H2 production in the ancient membrane-bound hydrogenase (Mbh) from the thermophilic archaeon Pyrococcus furiosus. Distinct from other hydrogenases, Mbh not only produces H2, but it couples this activity with ion transport across a membrane that powers the archaeal energy metabolism. Our study elucidates key mechanistic principles underlying H2 production and shed light on energy transducing enzymes that led to the evolution of modern mitochondrial respiratory enzymes.

biochemistry↗

bioRxiv2022

Effective Molecular Dynamics from Neural-Network Based Structure Prediction Models

Recent breakthroughs in neural-network based structure prediction methods, such as AlphaFold2 and RoseTTAFold, have dramatically improved the quality of computational protein structure prediction. These models also provide statistical confidence scores that can estimate uncertainties in the predicted structures, but it remains unclear to what extent these scores are related to the intrinsic conformational dynamics of proteins. Here we compare AlphaFold2 prediction scores with >60 s of explicit molecular dynamics simulations of 28 one- and two-domain proteins with varying degree of flexibility. We demonstrate a strong correlation between the statistical prediction scores and the explicit motion derived from extensive atomistic molecular dynamics simulations, and further derive an elastic network model based on the statistical scores of AlphFold2 (AF-ENM), which we benchmark in combination with coarse-grained molecular dynamics simulations. We show that our AF-ENM method reproduces the global protein dynamics with improved accuracy, providing a powerful way to derive effective molecular dynamics using neural-network based structure prediction models.

biophysics↗

bioRxiv2022

Structural basis of mammalian Complex IV inhibition by steroids

The mitochondrial electron transport chain maintains the proton motive force that powers ATP synthesis. The energy for this process comes from oxidation of NADH and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K- and D-pathways during turnover. The former is responsible for transferring two protons to the enzymes catalytic site upon reduction of the site, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid-binding can regulate CIV activity little is known about how this regulation occurs. Here we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the high-speed equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K-pathway. Single particle cryo-EM of CIV with glyco-diosgenin reveals a previously undescribed steroid-binding site adjacent to the K-pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle. SIGNIFICANCE STATEMENTMammalian complex IV (CIV), the final complex of the mitochondrial electron transport chain, uses electrons from cytochrome c to reduce oxygen to water, driving aerobic life. Although CIV functions as the main site for respiratory regulation, there is little structural or biochemical information on how this regulation occurs. Previous studies provided evidence of CIV regulation by steroids, but the steroid binding site and regulatory mechanism remain unclear. Using single particle cryogenic electron microscopy, we discover the binding site of the steroid-derived detergent, glyco-diosgenin. Results from flash photolysis kinetic experiments with CIV in the presence of glyco-diosgenin and cholesterol hemisuccinate are combined with cryo-EM and molecular simulations to elucidate how steroid binding limits proton uptake by the complex.

biophysics↗