Fig 2 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
(A) Iron-sulphur clusters in the hydrophilic domain of complex I from T. thermophilus. Edge-toedge distances are displayed. (B) The environment of N2, the final electron accepting iron-sulphur cluster. (C) Water-protein-based connection from the bulk to His169 shown with an orange mesh. Water molecules are shown with purple spheres. A highly conserved D408 (Nqo4 subunit) is a part of the connection.
Source publication
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 t...
Contexts in source publication
Context 1
... place when the structure of the hydrophilic domain of the enzyme was resolved using X-ray crystallography in 2006 at a resolution of 3.3 Å [51]. It revealed an intricate arrangement of FeS clusters and the electron transfer (eT) path from the FNM (flavin mononucleotide) to the terminal electron acceptor, the N2 FeS cluster (see Section 2.2.2 and Fig. 2). Later on, using state-of-the-art protein purification techniques and X-ray crystallography, research groups of Leonid Sazanov and Ulrich Brandt solved the structures of bacterial and eukaryotic complexes, respectively [32,35]. However, another key milestone was achieved in 2013, when the entire structure of complex I from Thermus ...
Context 2
... eT machinery in complex I is unique in that it resides in the hydrophilic domain of the enzyme in its entirety. This is different from all other respiratory and photosynthetic complexes, which harbour redox cofactors bound to the membrane subunits. Fig. 2 shows the eT path in complex I, which spans 70-80 Å. The X-ray data revealed the possible binding modes of NADH, next to the first electron acceptor; flavin mononucleotide (FMN) [57], stabilized by stacking interactions. The vicinity (~3.4 Å) of donor and acceptor suggests an adiabatic electron transfer with low driving force (E m,7 of ...
Context 3
... transfer with low driving force (E m,7 of NAD + / NADH ~ −320 mV and FMN/FMNH 2 ~ −300 mV). Electrons transferred to FMN from NADH via a rapid hydride (H − ) transfer [58] are subsequently passed on to the one-electron carriers, the FeS clusters. There are about 7-9 FeS clusters found in complex I, which are of the type; 2Fee2S or 4Fee4S ( Fig. 2A). It is well-known that the electronic properties of FeS are strongly perturbed by the surrounding protein, as shown in recent ultra-high resolution structural study ...
Context 4
... chain of complex I [54], which has been verified by the experiments [60,61]. Upon oxidation of FMN (in E. coli enzyme), the two electrons end up on two high-potential FeS clusters, one on N1a cluster, which is segregated from the main eT pathway, and the other on the N2 cluster, which is the direct electron donor to bound ubiquinone or quinone (Fig. 2) Though, in mammalian enzyme, this electron bifurcation is not observed, owing to apparently different redox potentials of the FeS clusters (N2 and ...
Context 5
... to N2) occurs in sub-millisecond timescales via pure electron tunneling with no tight coupling to proton pumping, which occurs an order of magnitude slower and also without any significant conformational changes, suggesting that the electron transfer through FeS chain is not directly linked to energy transduction. The long chain of metal centers (Fig. 2) serves what purpose, remains currently unknown, but it may be necessary to make eT to Q kinetically efficient, and maintaining low-levels of ROS production at the FMN site [62]. Moreover, it could be advantageous to segregate the ROS producing FMN center from the N2/Q redox chemistry for reasons not yet fully understood. Computational ...
Context 6
... whereas upstream eT (NADH to N2) will be briefly mentioned, even though the latter may be physiologically important. N2, a Fe 4 S 4 cluster, has a total charge of −2 or − 3 (including cysteinate ligands), when oxidized or reduced, respectively. However, structural data show that the cluster is surrounded by conserved positively charged residues (Fig. 2B), which would result in a total charge of the region to be neutral when oxidized, and −1 when reduced. Interestingly, a highly conserved residue His169 from Nqo4 subunit has been suggested to act as a redox-Bohr group [66], which would suggest that upon reduction, the charge of the region may stay neutral due to proton uptake from the ...
Context 7
... [66], which would suggest that upon reduction, the charge of the region may stay neutral due to proton uptake from the N-side of the membrane. When fluctuations are induced in the crystal structure by means of MD simulations, water molecules and polar amino acid residues are found to mediate a connectivity between the bulk N-phase and His169 (Fig. 2C). Such a path may be responsible for the reduction (of N2) coupled protonation of His169, supporting its role as a redox-Bohr group [66]. However, the recent data from Hyperfine EPR spectroscopy reveal that His169, though protonatable upon reduction of N2 cluster, is not a strong redoxBohr group, and the coupling between electron and ...
Similar publications
Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deacti...
The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the re...
Citations
... Complex II (succinate dehydrogenase) obtains electrons from succinate and transfers them to CoQ as well. CoQ then transfers the electrons it received from these two complexes to complex III (coenzyme Q), and ultimately to oxygen (Haapanen and Sharma, 2018). Oxygen molecules capture high-energy electrons at the flavin mononucleotide (FMN) of complex 1, leading to the formation of superoxide radicals. ...
The tumor microenvironment affects the structure and metabolic function of mitochondria in tumor cells. This process involves changes in metabolic activity, an increase in the amount of reactive oxygen species (ROS) in tumor cells compared to normal cells, the production of more intracellular free radicals, and the activation of oxidative pathways. From a practical perspective, it is advantageous to develop drugs that target mitochondria for the treatment of malignant tumors. Such drugs can enhance the selectivity of treatments for specific cell groups, minimize toxic effects on normal tissues, and improve combinational treatments. Mitochondrial targeting agents typically rely on small molecule medications (such as synthetic small molecules agents, active ingredients of plants, mitochondrial inhibitors or autophagy inhibitors, and others), modified mitochondrial delivery system agents (such as lipophilic cation modification or combining other molecules to form targeted mitochondrial agents), and a few mitochondrial complex inhibitors. This article will review these compounds in three main areas: oxidative phosphorylation (OXPHOS), changes in ROS levels, and endogenous oxidative and apoptotic processes.
... Thus, selecting molecular catalysts with suitable redox potential (between −0.45 and −0.7 V versus SHE since polysulfide electrochemical reduction without molecular catalyst usually occurs between −0.8 and −0.9 V versus SHE) and high redox kinetics is critical for the effectiveness of this approach. in a submillisecond timescale 26 . Flavin mononucleotide (riboflavin-5′-phosphate, FMN) is one of the most common electron shuttles that transfer the electrons from NADH to iron-sulfur cluster proteins, accelerating the cellular process including energy production 27 . ...
Aqueous redox flow battery (RFB) is one of the most promising technologies for grid-scale energy storage systems. Polysulfides are particularly attractive active materials owing to their low cost and high capacity, but the low energy efficiency and low operating current density limit their practical applications. Here we report an active and durable molecule catalyst, riboflavin sodium phosphate (FMN-Na), to transform sluggish polysulfide reduction reactions to fast redox reactions of FMN-Na via homogeneous catalysis. The FMN-Na catalyst substantially reduces the overpotential of a polysulfide–ferrocyanide RFB (S-Fe RFB) from more than 800 mV to 241 mV at 30 mA cm⁻². A catalysed S-Fe flow cell was demonstrated for 2,000 cycles at 40 mA cm⁻² with a low decay rate of 0.00004% per cycle (0.0017% per day). A catalysed polysulfide–iodide RFB operated for 1,300 cycles under 40 mA cm⁻² without capacity decay. This work addresses the bottleneck of polysulfide-based RFBs for long-duration energy storage applications.
... This long range proton transfer in the membrane arm of complex I can be driven by protons injected from E channel as part of Q redox chemistry. 10,28 Methods Hybrid quantum mechanical/molecular mechanical (QM/MM) simulations were performed on the high-resolution cryo-EM structure of mitochondrial complex I from Yarrowia lipolytica (PDB 7O71). The model system was limited to 6 core subunits: ND1, ND2, ND3, ND4, ND4L, ND6. ...
Respiratory complex I is a redox-driven proton pump contributing to about 40% of total proton motive force required for mitochondrial ATP generation. Recent high-resolution cryo-EM structural data revealed the positions of several water molecules in the membrane domain of the large enzyme complex. However, it remains unclear how protons flow in the membrane-bound antiporter-like subunits of complex I. Here, we performed multiscale computer simulations on high-resolution structural data to model explicit proton transfer processes in the ND2 subunit of complex I. Our results show protons can travel the entire width of antiporter-like subunits, including at the subunit-subunit interface, parallel to the membrane. We identify a previously unrecognized role of conserved tyrosine residues in catalyzing horizontal proton transfer, and that long-range electrostatic effects assist in reducing energetic barriers of proton transfer dynamics. Results from our simulations warrant a revision in several prevailing proton pumping models of respiratory complex I.
... Thus, by 2016 we knew the structures of bacterial and mammalian complex I, allowing for a range of hypotheses on the coupling mechanism to be developed in the subsequent years. These were mostly based on the long-range conformational changes due to a large distance (up to 200 Å) between electron transfer reactions (happening in the peripheral arm) and proton translocation across the membrane arm [17][18][19][20][21][22][23][24]. The proposals varied in many details in the absence of hard experimental evidence (high-resolution structures with resolved waters) on what happens with enzyme during turnover. ...
My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant 'black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm ('domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues.
... [29] Rotenone binds to complex I, thereby inhibiting electron flow and hence ultimately resulting in ROS accumulation. [30] Cells expressing hClpP-D92DiazK and hClpP-K261DiazK, which both previously showed the highest number of enriched proteins, were incubated for 6 hours with rotenone (1 μM) prior to work up (subsequently denoted as hClpP rot -D92DiazK and hClpP rot -K261DiazK, respectively). Proteomic workflow was conducted as described before and rotenone-treated and -untreated sample sets were compared for emergence of unique, rotenone-dependent proteins. ...
Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Site‐specific incorporation of the diazirine‐bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV‐irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis. In addition to confirming many of the previously annotated hClpP substrates, our approach adds a diverse set of new proteins to the hClpP interactome. Importantly, our workflow allows identifying substrate dynamics upon application of external cues in an unbiased manner. Identification of unique hClpP‐substrate proteins upon induction of mt oxidative stress, suggests that hClpP counteracts oxidative stress by processing of proteins that are involved in respiratory chain complex synthesis and maturation as well as in catabolic pathways.
... Los protones liberados están en solución en el medio celular, mientras que los grupos hidruro no, por ser sumamente reactivos. Por lo tanto, para fundamentar este mecanismo de reacción redox, se postula un complejo proceso de transferencia al NAD sin pasar por un imposible estado de hidruro libre en solución (Birrell y Hirst, 2013;Haapanen y Sharma, 2018). ...
This PhD thesis presents an extensive analysis of the results obtained in a research regarding the teaching as well as the learning concepts of Biological Chemistry implied in the cellular respiration topic, in eukaryotes. To carry out the present study, both quantitative and qualitative research methods were implemented. We worked, mainly, with three populations: one of secondary school level and two of university level, from the Common Basic Cycle (first year of university) and from the Faculty of Exact and Natural Sciences, both from the UBA.
Didactical, historical, and epistemological resources have been used as multidisciplinary theoretical frameworks to encompass visions of communication between experts and novices. Models, arguments, languages, and the evolution of scientific knowledge have been premises for the analysis of explanatory discourses on the analogy between chemical combustion and the global reaction of cellular respiration, and on the chain of electron transport in mitochondria. Likewise, those theoretical frameworks have also been used to support the design of original inquiry devices, which were applied to reveal learning efficiencies either in freshmen and fourth-year university students.
In Part A of the Thesis, the design of an original interdisciplinary proposal for teaching the concept of chemical combustión is presented. Its application on a secondary-level students population shows very satisfactory results, at the same time that reveals implicit underlying obstacles for professors as challenges from innovative teaching methodologies.
In Part B, a historical-epistemological analysis of the analogy between cellular respiration and glucose combustion and its use as a teaching device are carried out. Finally, freshmen students’ learning epistemological obstacles have been detected due to the application of this analogy during teaching processes.
In Part C of the Thesis, a historical-epistemological analysis of redox aspects of cellular respiration in mitochondria from the 18th century to the present is presented, with particular emphasis on the analysis of the Electron Transport Chain model, its structural components, and the participation of cofactors. Likewise, the research about the impact of teaching with the analogy finds new epistemological obstacles, particularly in freshmen’s learnings.
In Part D of the Thesis, a teaching proposal is presented to show the transport of electrons in prokaryotic cellular respiration, through an experimental device known as Sedimentary Microbial Fuel Cell that simulates an aerobic respiration mechanism for anaerobic bacteria (that do not have mitochondria). This PhD Thesis makes original historical-epistemological-didactic contributions in two dimensions, one related to the integrated visions about teaching redox aspects of cellular respiration, and the other to the design of original instruments. However these instruments are used in the present context to the inquiry about learning, they may also be used as teaching devices, due to their possible didactic power to elicit metacongintive reflections and help students to overcome conceptual obstacles during
their respective learning processes.
... Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the largest and most intricate membrane protein complex of the respiratory chain (1)(2)(3)(4). Complex I couples the transfer of electrons from reduced nicotinamide adenine dinucleotide (NADH) to ubiquinone with the translocation of protons across the inner mitochondrial membrane, with a stoichiometry of 4H + per NADH (5)(6)(7)(8). Redox-linked proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. ...
... The dynamics of complex I have been studied by molecular simulations (4,24,25) and by determining its structure in different functional states (16,(26)(27)(28). For mammalian and plant complex I, a change of the relative orientation of the peripheral arm and membrane arm was found in conjunction with well-defined changes in several central subunits (16,19,27,29). ...
... On the basis of these considerations, we propose a molecular mechanism of proton pumping in complex I, which resembles the well-documented proton pumping mechanism of cytochrome c oxidase, the terminal enzyme of the respiratory chain [ (43,44), see also (4) and (24)]. In cytochrome c oxidase, for every electron transfer to the oxygen reduction site, a proton is transferred to the protonloading site, followed by substrate proton transfer to the active site, which then electrostatically drives the ejection of the pumped proton from the proton-loading site to the P side of the membrane. ...
Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1-MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined, but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1-Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ~100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4-Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamic simulations, we define details of the proton translocation pathways and offer insights into the redox-coupled proton pumping mechanism of complex I.
... Complex I provides examples of both simple and complex proton transfer pathways. There are four proton paths, three through the antiporter subunits and one through the E-channel (Hirst, 2013;Ripple et al., 2013;Sazanov, 2015;Di Luca et al., 2017;Haapanen and Sharma, 2018;Saura and Kaila, 2019). The crystal structures show likely, linear paths through each antiporter subunit (Efremov and Sazanov, 2011;Baradaran et al., 2013;Zickermann et al., 2015) which have chain of well conserved acidic and basic residues in the center running parallel to the membrane ( Figures 8A,B) (Fearnley and Walker, 1992;Torres-Bacete et al., 2007;Efremov and Sazanov, 2012). ...
Biological membranes are barriers to polar molecules, so membrane embedded proteins control the transfers between cellular compartments. Protein controlled transport moves substrates and activates cellular signaling cascades. In addition, the electrochemical gradient across mitochondrial, bacterial and chloroplast membranes, is a key source of stored cellular energy. This is generated by electron, proton and ion transfers through proteins. The gradient is used to fuel ATP synthesis and to drive active transport. Here the mechanisms by which protons move into the buried active sites of Photosystem II (PSII), bacterial RCs (bRCs) and through the proton pumps, Bacteriorhodopsin (bR), Complex I and Cytochrome c oxidase (CcO), are reviewed. These proteins all use water filled proton transfer paths. The proton pumps, that move protons uphill from low to high concentration compartments, also utilize Proton Loading Sites (PLS), that transiently load and unload protons and gates, which block backflow of protons. PLS and gates should be synchronized so PLS proton affinity is high when the gate opens to the side with few protons and low when the path is open to the high concentration side. Proton transfer paths in the proteins we describe have different design features. Linear paths are seen with a unique entry and exit and a relatively straight path between them. Alternatively, paths can be complex with a tangle of possible routes. Likewise, PLS can be a single residue that changes protonation state or a cluster of residues with multiple charge and tautomer states.
... However, it appears that proton pumping is not associated with electron transfer along the Fe-S cluster chain. Rather, the redox state of the quinone substrate has been suggested to be the key factor [73]. The Krebs cycle enzyme and respiratory complex II, succinate dehydrogenase (SDH), is a prototypical multi-subunit Fe-S flavoenzyme along with the closely related FumR that catalyses the reverse reaction. ...
Iron-sulfur (Fe-S) flavoproteins form a broad and growing class of complex, multi-domain and often multi-subunit proteins coupling the most ancient cofactors (the Fe-S clusters) and the most versatile coenzymes (the flavin coenzymes, FMN and FAD). These enzymes catalyse oxidoreduction reactions usually acting as switches between donors of electron pairs and acceptors of single electrons, and vice versa. Through selected examples, the enzymes' structure-function relationships with respect to rate and directionality of the electron transfer steps, the role of the apoprotein and its dynamics in modulating the electron transfer process will be discussed.
... Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the largest and most intricate membrane protein complex of the respiratory chain (Galemou Yoga et al., 2020a;Haapanen and Sharma, 2018;Hirst, 2013;Sazanov, 2015). Complex I couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the inner mitochondrial membrane, with a stoichiometry of 4 H + per NADH (Galkin et al., 2006;Galkin et al., 1999;Jones et al., 2017;Wikström, 1984). ...
Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ~100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.
