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Complex I is a key proton-pumping enzyme in bacterial and mitochondrial respiratory electron transport chains. Using quantum chemistry and electrostatic calculations, we have examined the pKa of the reduced quinone QH-/QH2 in the catalytic cavity of complex I. We find that pKa (QH-/QH2) is very high, above 20. This means that the energy of a single protonation reaction of the doubly reduced quinone (i.e. the reduced semiquinone QH-) is sufficient to drive four protons across the membrane with a potential of 180 mV. Based on these calculations, we propose a possible scheme of redox-linked proton pumping by complex I. The model explains how the energy of the protonation reaction can be divided equally among four pumping units of the pump, and how a single proton can drive translocation of four additional protons in multiple pumping blocks.

Proton translocation across the cytoplasmic membrane is a vital process for all organisms. Dehalococcoides strains are strictly anaerobic organohalide respiring bacteria that lack quinones and cytochromes but express a large membrane-bound protein complex (OHR complex) proposed to generate a proton gradient. However, its functioning is unclear. By using a dehalogenase-based enzyme activity assay with deuterium-labelled water in various experimental designs, we obtained evidence that the halogen atom of the halogenated electron acceptor is substituted with a proton from the cytoplasm. This suggests that the protein complex couples exergonic electron flux through the periplasmic subunits of the OHR complex to the endergonic transport of protons from the cytoplasm across the cytoplasmic membrane against the proton gradient to the halogenated electron acceptor. Using computational tools, we located two proton-conducting half-channels in the AlphaFold2-predicted structure of the OmeB subunit of the OHR complex, converging in a highly conserved arginine residue that could play a proton gatekeeper role. The cytoplasmic proton half-channel in OmeB is connected to a putative proton-conducting path within the reductive dehalogenase subunit. Our results indicate that the reductive dehalogenase and its halogenated substrate serve as both electron and proton acceptors, providing insights into the proton translocation mechanism within the OHR complex and contributing to a better understanding of energy conservation in D. mccartyi strains. Our results reveal a very simple mode of energy conservation in anaerobic bacteria, showing that proton translocation coupled to periplasmic electron flow might have importance also in other microbial processes and biotechnological applications.

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli , resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis. [ABSTRACT FROM AUTHOR]

Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit photosystem II complex which functions in oxygenic photosynthesis, there is presently no consensus on the function of b-559 in the photosynthetic electron transport chain. The inability in earlier times to define a redox-linked function of this cytochrome was, to a large extent, a consequence of an absence of biochemical and structure information to complement an extensive array of spectrophotometric studies of the cytochrome in situ. Based on the location of hetero-dimeric b-559 in the photosystem II reaction center complex, derived from crystal crystallographic structure analysis, and the absence of a necessary redox function for the cytochrome in PSII, it is proposed that the main function of cytochrome b-559 is linked to its role as a structure component in the PSII reaction center complex. This function resides in the association of b-559 through its heme histidine residues in the trans-membrane domains of the PsbE and PsbF subunits of the PSII reaction center. These subunits, along with PsbJ, are inferred, from the analysis of structure, to define the intra-membrane portal in the PSII reaction center for plastoquinol (PQH2) export which, through the PSII complex, provides the redox link to the cytochrome b6f complex in the electron transfer chain. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in mitochondria. It catalyzes the four-electron reduction of O 2 to H 2 O and harnesses the redox energy to drive unidirectional proton translocation against a proton electrochemical gradient. A great deal of research has been conducted to comprehend the molecular properties of CcO. However, the mechanism by which the oxygen reduction reaction is coupled to proton translocation remains poorly understood. Here, we review the chemical properties of a variety of key oxygen intermediates of bovine CcO (bCcO) revealed by time-resolved resonance Raman spectroscopy and the structural features of the enzyme uncovered by serial femtosecond crystallography, an innovative technique that allows structural determination at room temperature without radiation damage. The implications of these data on the proton translocation mechanism are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer. [ABSTRACT FROM AUTHOR]

In this article, we develop a mathematical model for the rotary bacterial flagellar motor (BFM) based on the recently discovered structure of the stator complex (MotA5MotB2). The structure suggested that the stator also rotates. The BFM is modeled as two rotating nano-rings that interact with each other. Specifically, translocation of protons through the stator complex drives rotation of the MotA pentamer ring, which in turn drives rotation of the FliG ring in the rotor via interactions between the MotA ring of the stator and the FliG ring of the rotor. Preliminary results from the structure-informed model are consistent with the observed torque-speed relation. More importantly, the model predicts distinctive rotor and stator dynamics and their load dependence, which may be tested by future experiments. Possible approaches to verify and improve the model to further understand the molecular mechanism for torque generation in BFM are also discussed. [ABSTRACT FROM AUTHOR]

In this article, we develop a mathematical model for the rotary bacterial flagellar motor (BFM) based on the recently discovered structure of the stator complex (MotA5MotB2). The structure suggested that the stator also rotates. The BFM is modeled as two rotating nano-rings that interact with each other. Specifically, translocation of protons through the stator complex drives rotation of the MotA pentamer ring, which in turn drives rotation of the FliG ring in the rotor via interactions between the MotA ring of the stator and the FliG ring of the rotor. Preliminary results from the structure-informed model are consistent with the observed torque-speed relation. More importantly, the model predicts distinctive rotor and stator dynamics and their load dependence, which may be tested by future experiments. Possible approaches to verify and improve the model to further understand the molecular mechanism for torque generation in BFM are also discussed.

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the Escherichia coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.

The mutation rate of mitochondrial DNA (mtDNA) is 17 times higher than nuclear DNA, and these mutations can cause mitochondrial disease in 1 of 10.000 people. The T10609C mutation was identified in type 2 diabetes mellitus (T2DM) patients and the C10676G mutation in cataract patients, with both mutations occurring in the ND4L gene of mtDNA that encodes ND4L protein. ND4L protein, a subunit of complex I in the respiratory complex, has been shown to play a role in the proton translocation process. The purpose of this study was to investigate the effect of both mutations on the proton translocation mechanism. Mutation mapping showed changes in amino acids M47T (T10609C) and C69W (C10676G). The 100 ns molecular dynamics (MD) simulations performed on native and mutants of ND4L-ND6 subunits. It is revealed that the native model had a similar proton translocation pathway to that of complex I from other organisms. Interestingly, the mutant M47T and C69W showed the interruption of the translocation pathway by a hydrogen bond formation between Glu34 and Tyr157. It is observed that the mutations were restricting the passage of water molecules through the transmembrane region. These results could help to develop the computational assay for the validation of a specific genetic biomarker for T2DM and cataracts. [ABSTRACT FROM AUTHOR]

Proton translocation across the cytoplasmic membrane is a vital process for all organisms. Dehalococcoides strains are strictly anaerobic organohalide respiring bacteria that lack quinones and cytochromes but express a large membrane-bound protein complex (OHR complex) proposed to generate a proton gradient. However, its functioning is unclear. By using a dehalogenase-based enzyme activity assay with deuterium-labelled water in various experimental designs, we obtained evidence that the halogen atom of the halogenated electron acceptor is substituted with a proton from the cytoplasm. This suggests that the protein complex couples exergonic electron flux through the periplasmic subunits of the OHR complex to the endergonic transport of protons from the cytoplasm across the cytoplasmic membrane against the proton gradient to the halogenated electron acceptor. Using computational tools, we located two proton-conducting half-channels in the AlphaFold2-predicted structure of the OmeB subunit of the OHR complex, converging in a highly conserved arginine residue that could play a proton gatekeeper role. The cytoplasmic proton half-channel in OmeB is connected to a putative proton-conducting path within the reductive dehalogenase subunit. Our results indicate that the reductive dehalogenase and its halogenated substrate serve as both electron and proton acceptors, providing insights into the proton translocation mechanism within the OHR complex and contributing to a better understanding of energy conservation in D. mccartyi strains. Our results reveal a very simple mode of energy conservation in anaerobic bacteria, showing that proton translocation coupled to periplasmic electron flow might have importance also in other microbial processes and biotechnological applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hellmold, Eberwein, Phan, Kümmel, Einsle, Deobald and Adrian.)

Lugdunin, a novel thiazolidine cyclopeptide, exhibits micromolar activity against methicillin‐resistant Staphylococcus aureus (MRSA). For structure–activity relationship (SAR) studies, synthetic analogues obtained from alanine and stereo scanning as well as peptides with modified thiazolidine rings were tested for antimicrobial activity. The thiazolidine ring and the alternating d‐ and l‐amino acid backbone are essential. Notably, the non‐natural enantiomer displays equal activity, thus indicating the absence of a chiral target. The antibacterial activity strongly correlates with dissipation of the membrane potential in S. aureus. Lugdunin equalizes pH gradients in artificial membrane vesicles, thereby maintaining membrane integrity, which demonstrates that proton translocation is the mode of action (MoA). The incorporation of extra tryptophan or propargyl moieties further expands the diversity of this class of thiazolidine cyclopeptides. [ABSTRACT FROM AUTHOR]

Abstract Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (Vr H+-PPase), the Vr H+-PPase–2P i complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three Vr H+-PPases complexes (P i -, 2P i - and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2P i -bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among Vr H+-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of Vr H+-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer. Graphical Abstract Unlabelled Image Highlights • The pore diameters along the ion translocation pathway of Vr H+-PPase of different functional states were determined. • Pyrophosphate hydrolysis and proton pumping coupling efficacies of Vr H+-PPase mutants identify the roles of hydrophobic gate and exit channel. • The solvation of the hydrophobic gate of Vr H+-PPase was observed during the proton translocation. • Salt-bridge, E225–R562 at the exit channel acts as a latch to regulate ion release of Vr H+-PPase. [ABSTRACT FROM AUTHOR]

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson's and Alzheimer's, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping. [ABSTRACT FROM AUTHOR]

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Cytochrome c oxidase (COX) represents the main oxygen acceptor for respiration of aerobic organisms. The energy of respiration is stored via oxidative phosphorylation (OxPhos) in ATP, the general energy intermediate in living cells, or released as heat. During evolution from bacteria to mammals the complexity of OxPhos regulation increased at COX by increasing the number of subunits, by expression of subunit isoforms which are specific for tissues, developmental stages, and oxygen concentrations, and by reversible phosphorylation. The essential function of the “supernumerary” subunits became evident in patients with mitochondrial disease related to COX-deficiency, based on mutations in these subunit genes. While the basic regulation of energy transduction in OxPhos was explained by the Mitchell theory, an additional regulation of respiration and ATP synthesis was discovered in eukaryotic organisms, based on reversible and membrane potential-independent inhibition of COX activity at high ATP/ADP ratios. So far, only some of the complex regulatory functions of nuclear-encoded subunits were uncovered. [ABSTRACT FROM AUTHOR]

Plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a crucial enzyme that exists on the tonoplast to maintain pH homeostasis across the vacuolar membrane. This enzyme generates proton gradient between cytosol and vacuolar lumen by hydrolysis of a metabolic byproduct, pyrophosphate (PP i ). The regulation of V-PPase at protein level has drawn attentions of many workers for decades, but its mechanism is still unclear. In this work, we show that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14-3-3 isoforms were analyzed for their association with AVP1. In the presence of 14-3-3ν, -µ, -ο, and -ι, both enzymatic activities and its associated proton pumping of AVP1 were increased. Among these 14-3-3 proteins, 14-3-3 µ shows the highest stimulation on coupling efficiency. Furthermore, 14-3-3ν, -µ, -ο, and -ι exerted protection of AVP1 against the inhibition of suicidal substrate PP i at high concentration. Moreover, the thermal profile revealed the presence of 14-3-3ο improves the structural stability of AVP1 against high temperature deterioration. Additionally, the 14-3-3 proteins mitigate the inhibition of Na+ to AVP1. Besides, the binding sites/motifs of AVP1 were identified for each 14-3-3 protein. Taken together, a working model was proposed to elucidate the association of 14-3-3 proteins with AVP1 for stimulation of its enzymatic activity. [ABSTRACT FROM AUTHOR]

Biological energy currency ATP is produced by F 1 F o -ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the β subunit of F 1 F o -ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F 1 and F o motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex. [Display omitted] • A complete structure of the human ATP synthase complex • ADP release occurs when the β subunit is in the open conformation • Water molecules are defined in the inlet and outlet proton half-channels • Clinically relevant mutations cause the instability of the complex Lai et al. determine the complete structures of human ATP synthase in three main rotational states and one substate, providing mechanistic insights into the structure and function of the ATP synthase complexes and laying a foundation for target-based drug discovery. [ABSTRACT FROM AUTHOR]

Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochrome bo3 from E. coli at single-molecule level. Liposomes with reconstituted cytochrome bo3 were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochrome bo3 in the membrane; in ∼70 % of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a ΔpH (in the presence of the K+ ionophore valinomycin), which was stable over at least ∼800 s. No rapid changes in ΔpH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E. coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li et al. J. Am. Chem. Soc. (2015) 137, 16055-16063). [ABSTRACT FROM AUTHOR]

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b. [ABSTRACT FROM AUTHOR]

The mitochondrial disease often associated with various illnesses in relation to the activity of cells metabolites and the synthesis of adenosine triphosphate (ATP), including alteration in the mitochondrial DNA. The mutation of m.9053G>A at the ATP6 gene was found in patients with type 2 diabetes mellitus (DM type 2) and cataract. Therefore, this mutation is predicted to be clinical features of the 2 diseases. ATP6 gene encodes protein subunit of ATPase6, a part of ATP synthase, which is important in the electron transfer and proton translocation in intracellular respiration system. This study aims to investigate the mutation effect of m.9053G>A at the ATP6 gene (S167N) to the structure and function of ATPase6 using bioinformatics method. The structure of ATPase6 was constructed using homology modeling method. The crystal structure of bovine's ATP synthase (Protein Data Bank ID 5FIL) was used as a template because of high sequence similarity (77%) and coverage (96%) of the input sequence. The effect of mutation was investigated at the proton translocation channel of ATPase6. It is predicted that the channel was disrupted due to changes in electrostatic potential from serine to asparagine. Furthermore, molecular docking suggests that water binding on the proton translocation channel in the S167N mutant was different from the wild type. The result of this study is hoped to be useful in the development of a new genetic marker for DM type 2 and cataract. [ABSTRACT FROM AUTHOR]

Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli , subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M. [ABSTRACT FROM AUTHOR]

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3β3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator's integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ~30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine. [ABSTRACT FROM AUTHOR]