Respiratory Complex III: A Bioengine with a Ligand-Triggered Electron-Tunneling Gating Mechanism.

Respiratory complex III (a.k.a., the bc1 complex) plays a key role in the electron transport chain in aerobic cells. The bc1 complex exhibits multiple unique electron tunneling (ET) processes, such as ET-bifurcation at the Qo site and movement of the Rieske domain. Moreover, we previously discovered that electron tunneling in the low potential arm of the bc1 complex is regulated by a key phenylalanine residue (Phe90). The main goal of the current work is to study the dynamics of the key Phe90 residue in the electron tunneling reaction between heme bL and heme bH as a function of the occupancy of the Qo and Qi binding sites in the bc1 complex. We simulated the molecular dynamics of four model systems of respiratory complex III with different ligands bound at the Qo and Qi binding sites. In addition, we calculated the electron tunneling rate constants between heme bL and heme bH along the simulated molecular dynamics trajectories. The binding of aromatic ligands at the Qo site induces a conformational cascade that properly positions the Phe90 residue, reducing the through-space ET distance from ∼7 to ∼5.5 Å and thus enhancing the electron transfer rate between the heme bL and the heme bH redox pair. Also, the binding of aromatic ligands at the Qi site induces conformational changes that stabilize the Phe90 conformational variation from ∼1.5 to ∼0.5 Å. Hence, our molecular dynamics simulation results show an on-demand two-step conformational connection between the occupancy of the Qo and Qi binding sites and the conformational dynamics of the Phe90 residue. Additionally, our dynamic electron tunneling results confirm our previously reported findings that the Phe90 residue acts as an electron-tunneling gate or switch, controlling the electron transfer rate between the heme bL and heme bH redox systems.

Computational Modeling for the Oxidation Reactions of the Cysteine Residues with the Superoxide and the Organic Radical Species.

The current computational study analyzes the oxidation reactions of the superoxide and hydroxyl radicals with cysteine residues due to their importance as natural targets to neutralize the harmful reactive oxygen species. Due to the high reactivity of the hydroxyl radicals with the surrounding environment, we also studied the oxidation reactions of organic radicals with cysteine. In addition, we explored the different reaction pathways between cysteine and the superoxide radicals in both anionic and protonated forms. All calculations were performed at the integrated quantum mechanical/molecular mechanical level in an explicit water box under periodic boundary conditions. Higher energy barriers were observed for the organic radicals than the hydroxyl radical, where the chemical nature of the organic radical and the branching pattern are the main factors contributing to the Gibbs energy barriers. The superoxide radical oxidation pathway exhibits a more complex nature due to the complicated interplay of various factors such as the underlying reaction mechanism, the involved oxidizing agent, the kinetic accessibility of the oxidation reaction, and the thermodynamics favorability of those oxidation reactions. We also examined the effect of the solvent-assisted hydrogen atom transfer on the different reaction barriers, which was found to be kinetically unfavorable.

Respiratory complex I: Bottleneck at the entrance of quinone site requires conformational change for its opening.

The structure of the entire respiratory complex I is now known at reasonably high resolution for many species - bacteria, yeast, and several mammals, including human. The structure reveals an almost 30 angstrom tunnel-like chamber for ubiquinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. Here we characterize the geometric bottleneck forming the entrance of the quinone reaction chamber. Computer simulations of quinone/quinol passage through the bottleneck suggest that in all structures available, from bacterial to human, this bottleneck is too narrow for the quinone or quinol to pass and that a conformational change is required to open the channel. Moreover, the bottleneck is too narrow even for isoprenoid tail free passage. The closed structure can be an artifact of the crystallization packing forces, low temperature, or other unnatural conditions occurring in the structural data acquisition procedure that affect this flexible part of the enzyme. Two of the helices forming the bottleneck are in direct contact with the subunit (ND3) that was recently demonstrated to be involved in conformational changes during the redox proton pumping cycle, which indicates flexibility of that part of the enzyme. We conclude that the published structures are all locked in the unfunctional states and do not represent correctly the functional enzyme; we discuss possible ways to open the structure in the context of possible mechanisms of the enzyme.

Computational Modeling of the Disulfide Cross-Linking Reaction.

Disulfide cross-linking is one of the fundamental covalent bonds that exist prevalently in many biological molecules that is involved in versatile functional activities such as antibody stability, viral assembly, and protein folding. Additionally, it is a crucial factor in various industrial applications. Therefore, a fundamental understanding of its reaction mechanism would help gain insight into its different functional activities. Computational simulation of the disulfide cross-linking reaction with hydrogen peroxide (H2O2) was performed at the integrated quantum mechanical/molecular mechanical (QM/MM) level of theory in a water box under periodic boundary conditions. A benchmarking study for the barrier height of the disulfide formation step was performed on a model system between methanethiol and methane sulfenic acid to determine, for the QM system, the best-fit density functional theory (DFT) functional/basis set combination that produces comparable results to a higher-level theory of the coupled-cluster method. Computational results show that the disulfide cross-linking reaction with H2O2 reagent can proceed through a one-step or a two-step pathway for the high pKa cysteines or two different pathways for the low pKa cysteines to ultimately produce the sulfenic acid/sulfenate intermediate complex. Subsequently, those intermediates react with another neutral/anionic cysteine residue to form the cysteine product. In addition, the solvent-assisted proton-exchange/proton-transfer effects were examined on the energetic barriers for the different transition states, and the molecular contributions of the chemically involved water molecules were studied in detail.

Mutations in NDUFS1 Cause Metabolic Reprogramming and Disruption of the Electron Transfer.

Complex I (CI) is the first enzyme of the mitochondrial respiratory chain and couples the electron transfer with proton pumping. Mutations in genes encoding CI subunits can frequently cause inborn metabolic errors. We applied proteome and metabolome profiling of patient-derived cells harboring pathogenic mutations in two distinct CI genes to elucidate underlying pathomechanisms on the molecular level. Our results indicated that the electron transfer within CI was interrupted in both patients by different mechanisms. We showed that the biallelic mutations in NDUFS1 led to a decreased stability of the entire N-module of CI and disrupted the electron transfer between two iron-sulfur clusters. Strikingly interesting and in contrast to the proteome, metabolome profiling illustrated that the pattern of dysregulated metabolites was almost identical in both patients, such as the inhibitory feedback on the TCA cycle and altered glutathione levels, indicative for reactive oxygen species (ROS) stress. Our findings deciphered pathological mechanisms of CI deficiency to better understand inborn metabolic errors.

Concerted Two-Electron Reduction of Ubiquinone in Respiratory Complex I.

Respiratory complex I catalyzes two-electron/two-proton reduction of a ubiquinone (Q) substrate bound at its Q-binding pocket; upon reduction, ubiquinole carries electrons further down the electron transport chain. The mechanism of this two-electron transfer reaction is poorly understood. Here we consider a hypothetical scheme in which two electrons transfer together with two protons in a concerted fashion. On one side, a coupled electron/proton transfer occurs from the reduced N2 FeS cluster and protonated His38 residue, respectively, while on the other side a hydrogen atom transfer occurs from the neutral Tyr87 residue, generating a tyrosyl radical. A method to evaluate the coupling matrix element that corresponds to a concerted tunneling of two electrons was developed. Overall, our calculations indicate that the concerted reaction is feasible, in which case a transient tyrosyl radical is formed during the catalytic cycle of the enzyme.

Polarizable Embedding for Excited-State Reactions: Dynamically Weighted Polarizable QM/MM.

Recent interest in polarizable embedding methods for electronic excited states has so far been focused on optical absorption and emission spectra calculations. To explore the suitability of these methods for excited-state reactions, we constructed a simple molecular system with an electronic crossing coupled to a polarizable species: the triatomic LiFBe. We found that current polarizable QM/MM methods inadequately describe the potential energy surfaces in this system, particularly close to the electronic crossing, so we developed a new polarizable embedding method called dynamically weighted polarizable QM/MM. The new method reproduces the potential energy surfaces of LiFBe from full-system multireference configuration interaction singles and doubles calculations with near-quantitative accuracy.

Electron tunneling in proteins program.

We developed a unique integrated software package (called Electron Tunneling in Proteins Program or ETP) which provides an environment with different capabilities such as tunneling current calculation, semi-empirical quantum mechanical calculation, and molecular modeling simulation for calculation and analysis of electron transfer reactions in proteins. ETP program is developed as a cross-platform client-server program in which all the different calculations are conducted at the server side while only the client terminal displays the resulting calculation outputs in the different supported representations. ETP program is integrated with a set of well-known computational software packages including Gaussian, BALLVIEW, Dowser, pKip, and APBS. In addition, ETP program supports various visualization methods for the tunneling calculation results that assist in a more comprehensive understanding of the tunneling process. © 2016 Wiley Periodicals, Inc.

Novel Inhibitors for a Novel Binding Site in Respiratory Complex III.

A new binding site and potential novel inhibitors of the respiratory complex III are described. The site is located at the opposite side of the enzyme with respect to ubiquinol binding site (Qo site), and distinctly different from both Qo and Qi sites (hence designated as Non-Q binding site, NQ). NQ site binding pocket extends up close to Phe90 residue, an internal switch (LH switch) that regulates electron transfer between heme bL and heme bH of the low potential redox chain. Docking studies and molecular dynamics simulations of different molecules to the NQ site revealed potential ligands which exhibit a novel inhibitory effect for bc1 complex by switching the LH switch to "off" conformation, thereby shutting down electron transfer in the low potential redox chain. Moreover, the novel inhibitors have lower binding affinity for both Qo and Qi sites, and hence do not interfere with binding of the natural ligands to those sites. The inhibitory activity of those novel ligands in bc1 complex is suggested to promote the production of reactive oxygen species (ROS) at the Qo site. Hence those ligands are potential candidates for designing new "mitocan" drugs.

Internal switches modulating electron tunneling currents in respiratory complex III.

In different X-ray crystal structures of bc1 complex, some of the key residues of electron tunneling pathways are observed in different conformations; here we examine their relative importance in modulating electron transfer and propose their possible gating function in the Q-cycle. The study includes inter-monomeric electron transfer; here we provide atomistic details of the reaction, and discuss the possible roles of inter-monomeric electronic communication in bc(1) complex. Binding of natural ligands or inhibitors leads to local conformational changes which propagate through protein and control the conformation of key residues involved in the electron tunneling pathways. Aromatic-aromatic interactions are highly utilized in the communication network since the key residues are aromatic in nature. The calculations show that there is a substantial change of the electron transfer rates between different redox pairs depending on the different conformations acquired by the key residues of the complex.

Quantum Calculations of Electron Tunneling in Respiratory Complex III.

The most detailed and comprehensive to date study of electron transfer reactions in the respiratory complex III of aerobic cells, also known as bc1 complex, is reported. In the framework of the tunneling current theory, electron tunneling rates and atomistic tunneling pathways between different redox centers were investigated for all electron transfer reactions comprising different stages of the proton-motive Q-cycle. The calculations reveal that complex III is a smart nanomachine, which under certain conditions undergoes conformational changes gating electron transfer, or channeling electrons to specific pathways. One-electron tunneling approximation was adopted in the tunneling calculations, which were performed using hybrid Broken-Symmetry (BS) unrestricted DFT/ZINDO levels of theory. The tunneling orbitals were determined using an exact biorthogonalization scheme that uniquely separates pairs of tunneling orbitals with small overlaps out of the remaining Franck-Condon orbitals with significant overlap. Electron transfer rates in different redox pairs show exponential distance dependence, in agreement with the reported experimental data; some reactions involve coupled proton transfer. Proper treatment of a concerted two-electron bifurcated tunneling reaction at the Q(o) site is given.

Transition Flux Formula for the Electronic Coupling Matrix Element.

The transition flux formula for the coupling matrix element of long-distance electron transfer reactions is discussed. Here we present a new derivation which is based on the Golden Rule approach. The electronic Franck-Condon factor that appears in the multielectronic formulation of the coupling element is discussed using the concept of tunneling time. An application of the tunneling flux theory to electron transfer reactions in a model system based on the low-potential heme and high-potential heme (heme bL)/(heme bH) redox pair of ubiquinol:cytochrome c oxidoreductase complex is described; the results are compared to those obtained by measuring energy splitting of the donor/acceptor multielectronic states and the direct calculation method.