Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.     

Hydrogen bond network analysis reveals the pathway for the proton transfer in the E-channel of T. thermophilus Complex I

Complex I, NADH-ubiquinone oxidoreductase, is the first enzyme in the mitochondrial and bacterial aerobic respiratory chain. It pumps four protons through four transiently open pathways from the high pH, negative, N-side of the membrane to the positive, P-side driven by the exergonic transfer of electrons from NADH to a quinone. Three protons transfer through subunits descended from antiporters, while the fourth, E-channel is unique. The path through the E-channel is determined by a network analysis of hydrogen bonded pathways obtained by Monte Carlo sampling of protonation states, polar hydrogen orientation and water occupancy. Input coordinates are derived from molecular dynamics trajectories comparing oxidized, reduced (dihydro) and no menaquinone-8 (MQ). A complex proton transfer path from the N- to the P-side is found consisting of six clusters of highly connected hydrogen-bonded residues. The network connectivity depends on the presence of quinone and its redox state, supporting a role for this cofactor in coupling electron and proton transfers. The N-side is more organized with MQ-bound complex I facilitating proton entry, while the P-side is more connected in the apo-protein, facilitating proton exit. Subunit Nqo8 forms the core of the E channel; Nqo4 provides the N-side entry, Nqo7 and then Nqo10 join the pathway in the middle, while Nqo11 contributes to the P-side exit.     

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?Å wide 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.     

The cytochrome b6f complex. Novel aspects

The Cyt b ₆ f complex from plant chloroplasts, the green alga Chlamydomonas reinhardtii , and the thermophilic cyanobacterium, Mastigocladus laminosus , can be isolated in a highly active state, in which it is dimeric and contains one bound chlorophyll a molecule per monomeric unit. The latter feature is a distinguishing trait compared to the b ₆ f complex of bacterial photosynthesis and the respiratory chain. In contrast to the trans-membrane domains of the b ₆ f complex, and of most other integral membrane proteins, which are characterized by an a -helical structure, the p -side peripheral domains, consisting of Cyt f and the Rieske protein, have a predominantly β-strand secondary structure motif. One consequence of this motif is an extension of these polypeptides from the membrane surface. For example, the length of Cyt f is 75 Å. The heme Fe is 45 Å from the α-carbon of Arg250 at the membrane bilayer interface and, even though Cyt f may be tilted relative to the membrane plane, the heme electron transfer reactions are carried out far from the membrane surface. The presence of an internal 5 water chain, which has the properties of a proton wire, with one water H-bonded to the histidine-25 heme ligand, also suggests that the pathway of long distance H⁺ translocation traverses the extended p -side protein domain of the b ₆ f complex. A mechanism of H⁺ transfer in the chain that is coupled to the redox state of the heme, in which a proton is transferred into the chain to compensate the extra electron in the ferro-heme, is proposed.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Epitopes of monoclonal antibodies which inhibit ubiquinol oxidase activity of Escherichia coli cytochrome d complex localize functional domain.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases: the cytochrome d complex and the cytochrome o complex. Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water. Both oxidases are coupling sites in the respiratory chain; electron transfer from ubiquinol to oxygen results in the generation of a proton electrochemical potential difference across the membrane. The cytochrome d complex is a heterodimer (subunits I and II) that has three heme prosthetic groups. Previous studies characterized two monoclonal antibodies that bind to subunit I and specifically block the ability of the enzyme to oxidize ubiquinol. In this paper, the epitopes of both of these monoclonal antibodies have been mapped to within a single 11-amino acid stretch of subunit I. The epitope is located in a large hydrophilic loop between the fifth and sixth putative membrane-spanning segments. Binding experiments with these monoclonal antibodies show this polypeptide loop to be periplasmic. Such localization suggests that the loop may be close to His186, which has been identified as one of the axial ligands of cytochrome b558. Together, these data begin to define a functional domain in which ubiquinol is oxidized near the periplasmic surface of the membrane.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK(a) of the Schiff base (the primary proton donor) and the low pK(a) of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Transhydrogenation reactions catalyzed by mitochondrial NADH-ubiquinone oxidoreductase (Complex I)

NADH-ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory electron transport chain in mitochondria. It conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the inner membrane. Complex I catalyzes NADPH oxidation, NAD+ reduction, and hydride transfers from reduced to oxidized nicotinamide nucleotides also. Here, we investigate the transhydrogenation reactions of complex I, using four different nucleotide pairs to encompass a range of reaction rates. Our experimental data are described accurately by a ping-pong mechanism with double substrate inhibition. Thus, we contend that complex I contains only one functional nucleotide binding site, in agreement with recent structural information, but in disagreement with previous mechanistic models which have suggested that two different binding sites are employed to catalyze the two half reactions. We apply the Michaelis-Menten equation to describe the productive states formed when the nucleotide and the active-site flavin mononucleotide have complementary oxidation states, and dissociation constants to describe the nonproductive states formed when they have the same oxidation state. Consequently, we derive kinetic and thermodynamic information about nucleotide binding and interconversion in complex I, relevant to understanding the mechanisms of coupled NADH oxidation and NAD+ reduction, and to understanding how superoxide formation by the reduced flavin is controlled. Finally, we discuss whether NADPH oxidation and/or transhydrogenation by complex I are physiologically relevant processes.     

A new model for the evolution of carnivory in the bladderwort plant (utricularia): adaptive changes in cytochrome C oxidase (COX) provide respiratory power

The evolution of carnivorous plants has been modeled as a selective tradeoff between photosynthetic costs and benefits in nutrient-poor habitats. Although possibly applicable for pitfall and flypaper trappers, more variables may be required for active trapping systems. Bladderwort (utricularia) suction traps react to prey stimuli with an extremely rapid release of elastic instability. Trap setting requires considerable energy to engage an active ion transport process whereby water is pumped out through the thin bladder walls to create negative internal pressure. Accordingly, empirical estimates have shown that respiratory rates in bladders are far greater than in leafy structures. Cytochrome C oxidase (COX) is a multi-subunit enzyme that catalyzes the respiratory reduction of oxygen to water and couples this reaction to translocation of protons, generating a transmembrane electrochemical gradient that is used for the synthesis of adenosine triphosphate (ATP). We have previously demonstrated that two contiguous cysteine residues in helix 3 of COX subunit I (COX I) have evolved under positive Darwinian selection. This motif, absent in approximately 99.9 % of databased COX I proteins from eukaryotes, Archaea, and Bacteria, lies directly at the docking point of COX I helix 3 and cytochrome C. Modeling of bovine COX I suggests the possibility that a vicinal disulfide bridge at this position could cause premature helix termination. The helix 3-4 loop makes crucial contacts with the active site of COX, and we postulate that the C-C motif might cause a conformational change that decouples (or partly decouples) electron transport from proton pumping. Such decoupling would permit bladderworts to optimize power output (which equals energy times rate) during times of need, albeit with a 20 % reduction in overall energy efficiency of the respiratory chain. A new model for the evolution of bladderwort carnivory is proposed that includes respiration as an additional tradeoff parameter.     

Crystal structures of the carboxyl terminal domain of rat 10-formyltetrahydrofolate dehydrogenase: implications for the catalytic mechanism of aldehyde dehydrogenases

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.     

Functional implications from an unexpected position of the 49-kDa subunit of NADH:ubiquinone oxidoreductase

Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.     

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.     

Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB

Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution and water-interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans-membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far.     

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

Elevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨ_m) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨ_m and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance.     

On the molecular mechanism of liver alcohol dehydrogenase: Monte Carlo simulations and X-ray studies of water in the substrate channel

Water structure in the substrate channel of liver alcohol dehydrogenase as a function of the oxidation state of the coenzyme nicotinamide ring has been studied with Monte Carlo simulations. X-ray data on water structure has been analyzed. The simulations show an order-disorder effect in the water distribution produced by the charge state of the ring; also, solvation-desolvation effects are detected. For positively charged ring, the water molecules form a fluctuated H-bonded network that connects the deeply buried active site zinc to the bulk solvent. This network together with the side chain of Ser-48 most likely is the support for a proton relay system thereby providing a mechanism responsible of the pKa shift of the zinc-bound water as it is produced by the oxidized coenzyme binding.     

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c‐di‐GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme‐substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c‐di‐GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c‐di‐GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c‐di‐GMP to promote the O3'? P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670–1680. © 2016 Wiley Periodicals, Inc.     

Crystal structures of type II restriction endonuclease EcoO109I and its complex with cognate DNA

EcoO109I is a type II restriction endonuclease that recognizes the DNA sequence of RGGNCCY. Here we describe the crystal structures of EcoO109I and its complex with DNA. A comparison of the two structures shows that the catalytic domain moves drastically to capture the DNA. One metal ion and two water molecules are observed near the active site of the DNA complex. The metal ion is a Lewis acid that stabilizes the pentavalent phosphorus atom in the transition state. One water molecule, activated by Lys-126, attacks the phosphorus atom in an S(N)2 mechanism, whereas the other water interacts with the 3'-leaving oxygen to donate a proton to the oxygen. EcoO109I is similar to EcoRI family enzymes in terms of its DNA cleavage pattern and folding topology of the common motif in the catalytic domain, but it differs in the manner of DNA recognition. Our findings propose a novel classification of the type II restriction endonucleases and lead to the suggestion that EcoO109I represents a new subclass of the EcoRI family.     

A Computational Model of Reactive Oxygen Species and Redox Balance in Cardiac Mitochondria

Elevated levels of reactive oxygen species (ROS) play a critical role in cardiac myocyte signaling in both healthy and diseased cells. Mitochondria represent the predominant cellular source of ROS, specifically the activity of complexes I and III. The model presented here explores the modulation of electron transport chain ROS production for state 3 and state 4 respiration and the role of substrates and respiratory inhibitors. Model simulations show that ROS production from complex III increases exponentially with membrane potential (ΔΨ_m) when in state 4. Complex I ROS release in the model can occur in the presence of NADH and succinate (reverse electron flow), leading to a highly reduced ubiquinone pool, displaying the highest ROS production flux in state 4. In the presence of ample ROS scavenging, total ROS production is moderate in state 3 and increases substantially under state 4 conditions. The ROS production model was extended by combining it with a minimal model of ROS scavenging. When the mitochondrial redox status was oxidized by increasing the proton permeability of the inner mitochondrial membrane, simulations with the combined model show that ROS levels initially decline as production drops off with decreasing ΔΨ_m and then increase as scavenging capacity is exhausted. Hence, this mechanistic model of ROS production demonstrates how ROS levels are controlled by mitochondrial redox balance.     

A tyrosine residue deprotonates during oxygen reduction by the caa3 reductase from Rhodothermus marinus

Heme-copper oxygen reductases catalyze proton translocation across the cellular membrane; this takes place during the reaction of oxygen to water. We demonstrate with attenuated total reflection-Fourier transform infrared (ATR-FTIR) difference spectroscopy that a tyrosine residue of the oxygen reductase from the thermohalophilic Rhodothermus marinus becomes deprotonated in the transition from the oxidized state to the catalytic intermediate ferryl state P(M). This tyrosine residue is most probably Y256, the helix VI tyrosine residue proposed to substitute for the D-channel glutamic acid that is absent in this enzyme. Comparison with the mitochondrial like oxygen reductase from Rhodobacter sphaeroides suggests that proton transfer from a strategically situated donor to the active site is a crucial step in the reaction mechanism of oxygen reductases.