Molecular dynamics and structural models of the cyanobacterial NDH-1 complex

NDH-1 is a gigantic redox-driven proton pump linked with respiration and cyclic electron flow in cyanobacterial cells. Based on experimentally resolved X-ray and cryo-EM structures of the respiratory complex I, we derive here molecular models of two isoforms of the cyanobacterial NDH-1 complex involved in redox-driven proton pumping (NDH-1L) and CO₂-fixation (NDH-1MS). Our models show distinct structural and dynamic similarities to the core architecture of the bacterial and mammalian respiratory complex I. We identify putative plastoquinone-binding sites that are coupled by an electrostatic wire to the proton pumping elements in the membrane domain of the enzyme. Molecular simulations suggest that the NDH-1L isoform undergoes large-scale hydration changes that support proton-pumping within antiporter-like subunits, whereas the terminal subunit of the NDH-1MS isoform lacks such structural motifs. Our work provides a putative molecular blueprint for the complex I-analogue in the photosynthetic energy transduction machinery and demonstrates that general mechanistic features of the long-range proton-pumping machinery are evolutionary conserved in the complex I-superfamily.     

Single particle analysis confirms distal location of subunits NuoL and NuoM in Escherichia coli complex I

Respiratory complex I catalyses the transfer of electrons from NADH to quinone coupled to the translocation of protons across the membrane. The mechanism of coupling and the structure of the complete enzyme are not known. The membrane domain of the complex contains three similar antiporter-like subunits NuoL/M/N, probably involved in proton pumping. We have previously shown that subunits NuoL/M can be removed from the rest of the complex, suggesting their location at the distal end of the membrane domain. Here, using electron microscopy and single particle analysis, we show that subunits NuoL and M jointly occupy a distal half of the membrane domain, separated by about 10nm from the interface with the peripheral arm. This indicates that coupling mechanism of complex I is likely to involve long range conformational changes.     

Proton motive function of the terminal antiporter-like subunit in respiratory complex I

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.     

Functional dissection of the proton pumping modules of mitochondrial complex I

Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.     

Projection structure of the membrane domain of Escherichia coli respiratory complex I at 8 A resolution

Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22(1)2(1). Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 A resolution. About 60 transmembrane alpha-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.     

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.     

The mechanism of proton translocation in respiratory complex I from molecular dynamics

Abstract

Respiratory complex I, the biggest enzyme of respiratory chain, plays a key role in energy production by the mitochondrial respiratory chain and has been implicated in many human neurodegenerative diseases. Recently, the crystal structure of respiratory complex I is reported. We perform 50?ns molecular dynamics simulations on the membrane domain of respiratory complex I under two hypothetical states (oxidized state and reduced state). We find that the density of water molecules in the trans-membrane domain under reduced state is bigger than that under oxidized state. The connecting elements (helix HL and β-hairpins-helix element) fluctuate stronger under reduced state than that under oxidized state, causing more internal water molecules and facilitating the proton conduction. The conformational changes of helix HL and the crucial charged residue Glu in TM5 play key roles in the mechanism of proton translocation. Our results illustrate the dynamic behavior and the potential mechanism of respiratory complex I, which provides the structural basis for drug design of respiratory complex I.     

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na⁺/H⁺ antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH 7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.     

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.     

A two-state stabilization-change mechanism for proton-pumping complex I

Despite its central function in oxidative phosphorylation, the molecular mechanism of proton pumping respiratory complex I is still elusive. In recent years, considerable progress has been made towards understanding structure/function relationships in this very large and complicated membrane protein complex. Last year X-ray crystallographic analysis of bacterial and mitochondrial complex I provided important insights into its molecular architecture. Based on this evidence, here a hypothetical molecular mechanism for redox-driven proton pumping of complex I is proposed. According to this mechanism, two pump modules are driven by two conformational strokes that are generated by stabilization of the anionic forms of semiquinone and ubiquinol that are formed in the peripheral arm of complex I during turnover. This results in the experimentally determined pumping stoichiometry of 4 H(+)/2e(-). In the two-state model, electron transfer from iron-sulfur cluster N2 is allowed only in the 'E-state,' while protonation of the substrate is only possible in the stabilizing 'P-state.' In the membrane arm, transition from the E- to the P-state drives the two pump modules via long range conformational energy transfer through the recently discovered helical transmission element connecting them. The proposed two-state stabilization-change mechanism is fully reversible and thus inherently explains the operation of complex I in forward and reverse mode. This article is part of a Special Issue entitled Allosteric cooperativity in respiratory proteins.     

A modeling and simulation perspective on the mechanism and function of respiratory complex I

Respiratory complex I is a giant redox-driven proton pump, and central to energy production in mitochondria and bacteria. It catalyses the reduction of quinone to quinol, and converts the free energy released into the endergonic proton translocation across the membrane. The proton pumping sets up the proton electrochemical gradient, which propels the synthesis of ATP. Despite the availability of extensive biochemical, biophysical and structural data on complex I, the mechanism of coupling between the electron and proton transfer reactions remain uncertain. In this work, we discuss current state-of-the-art in the field with particular emphasis on the molecular mechanism of respiratory complex I, as deduced from computational modeling and simulation approaches, but in strong alliance with the experimental data. This leads to novel synthesis of mechanistic ideas on a highly complex enzyme of the electron transport chain that has been associated with a number of mitochondrial and neurodegenerative disorders.     

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.     

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.     

Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.     

Tracing the trail of protons through complex I of the mitochondrial respiratory chain

Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes--the respiratory chain--that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.     

Toward a Characterization of the Connecting Module of Complex I

Complex I [NADH–ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB–NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.     

Complex I: A Chimaera of a Redox and Conformation-Driven Proton Pump?

From phylogenetic sequence analysis, it can be concluded that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) has evolved from preexisting modules for electron transfer and proton translocation. It is built up by a peripheral NADH dehydrogenase module, an amphipatic hydrogenase module, and a membrane-bound transporter module. These modules, or at least part of them, are also present in various other bacterial enzymes. It is assumed that they fulfill a similar function in complex I and related enzymes. Based on the function of the individual modules, it is possible to speculate about the mechanism of complex I. The hydrogenase module might work as a redox-driven proton pump, while the transporter module might act as a conformation-driven proton pump. This implies that complex I contains two energy-coupling sites. The NADH dehydrogenase module seems to be involved in electron transfer and not in proton translocation.     

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F₁F_o-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F_o domain, which is coupled to the ATP-producing F₁ domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F₁F_o-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.