Functional Dynamics of an Ancient Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) is a redox-driven Na+/H+ transporter that employs the energy from hydrogen gas (H2) production to catalyze proton pumping and Na+/H+ exchange across cytoplasmic membranes of archaea. Despite a recently resolved structure of this ancient energy-transducing enzyme [Yu et al. Cell 2018, 173, 1636-1649], the molecular principles of its redox-driven ion-transport mechanism remain puzzling and of major interest for understanding bioenergetic principles of early cells. Here we use atomistic molecular dynamics (MD) simulations in combination with data clustering methods and quantum chemical calculations to probe principles underlying proton reduction as well as proton and sodium transport in Mbh from the hyperthermophilic archaeon Pyrococcus furiosus. We identify putative Na+ binding sites and proton pathways leading across the membrane and to the NiFe-active center as well as conformational changes that regulate ion uptake. We suggest that Na+ binding and protonation changes at a putative ion-binding site couple to proton transfer across the antiporter-like MbhH subunit by modulating the conformational state of a conserved ion pair at the subunit interface. Our findings illustrate conserved coupling principles within the complex I superfamily and provide functional insight into archaeal energy transduction mechanisms.

Deactivation blocks proton pathways in the mitochondrial complex I.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

Design of buried charged networks in artificial proteins.

Soluble proteins are universally packed with a hydrophobic core and a polar surface that drive the protein folding process. Yet charged networks within the central protein core are often indispensable for the biological function. Here, we show that natural buried ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif from its surroundings to gain structural stability. To explore this effect, we build artificial proteins with buried ion-pairs by combining directed computational design and biophysical experiments. Our findings illustrate how perturbation in charged networks can introduce structural rearrangements to compensate for desolvation effects. We validate the physical principles by resolving high-resolution atomic structures of the artificial proteins that are resistant towards unfolding at extreme temperatures and harsh chemical conditions. Our findings provide a molecular understanding of functional charged networks and how point mutations may alter the protein's conformational landscape.

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton-electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics.

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200 Šwide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.