The coupling mechanism of mammalian respiratory complex I

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo-electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions

Mammalian mitochondrial complex I structure and disease causing mutations

Complex I has an essential role in ATP production by coupling electron transfer from NADH to quinone with translocation of protons across the inner mitochondrial membrane. Isolated complex I deficiency is a frequent cause of mitochondrial inherited diseases. Complex I has also been implicated in cancer, ageing, and neurodegenerative conditions. Until recently, the understanding of complex I deficiency on the molecular level was limited due to the lack of high-resolution structures of the enzyme. However, due to developments in single particle cryo-electron microscopy (cryo-EM), recent studies have reported nearly atomic resolution maps and models of mitochondrial complex I. These structures significantly add to our understanding of complex I mechanism and assembly. The disease-causing mutations are discussed here in their structural context

Structure and mechanism of mitochondrial proton-translocating transhydrogenase

Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers7,8,9

Clarifying the supercomplex: The higher-order organization of the mitochondrial electron transport chain

The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI-CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII2 and CIV (SC I+III2+IV, known as the respirasome), as well as with CIII2 alone (SC I+III2). CIII2 forms a supercomplex with CIV (SC III2+IV) and CV forms dimers (CV2). Recent cryo-EM studies have revealed the structures of SC I+III2+IV and SC I+III2. Furthermore, recent work has shed light on the assembly and function of the SCs. Here we review and compare these recent studies and discuss how they have advanced our understanding of mitochondrial electron transport

Cryo-EM grid optimization for membrane proteins

Cryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins

BETWEEN RUSSIA, CHINA, AND THE US: WHY DOES AUTHORITARIANISM PREVAIL IN KAZAKHSTAN?

Abstract The significance of the external influence of Russia and China on the strengthening of authoritarianism in Kazakhstan has yet to be revealed due to the lack of a theory of the interaction of external influence and domestic political regimes. In my research, I sought to show that the authoritarian regimes of the Eurasian states have a common political legacy that unites the states that have joined them into a single political world. Their main actors are the supporting states interested in preserving kindred regimes throughout the common political space. Using tracing process analysis, I have mapped out causal relationships through the survival mechanism of kinship regimes and restoration of interdependence between pivotal and non-pivotal actors that determine how key states, such as Russia and China, react to external factors—geopolitical threats. The most important conclusion of my research is that the weakening of an authoritarian regime in one of the non-pivotal states poses a threat to regimes in core states such as China and Russia and triggers their survival reaction through increased foreign policy towards non-core states. In addition, my research has shown that political regimes that share a common legacy are interdependent and that the external influence of the central states of a common political space is primary in maintaining internal regimes throughout its entire length. Therefore, pivotal states of other political legacies can influence political regime change when the causal mechanism fails

Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk

The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between “closed” and “open” conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs

Tax monitoring as an alternative to existing forms of tax control in russia

Introduction: The article deals with the questions of the tax control exercised by tax authorities. Tax control in tax system of any country takes the central place. Essence of tax control is motivation of taxpayers through system of tax sanctions to observance of the tax law and to timely and payment in full of tax payments. Except the main forms of tax control, such as off-site and on-site tax audit, since 2016 a new form of tax control - tax monitoring or "horizontal" monitoring joined into force. The article is devoted to consideration of prospects of tax monitoring. Methods: A methodological basis of research is the dialectic method of knowledge, system approach to the analysis of the considered facts and the phenomena. Research is based on application of methods of the analysis, system and complexity, the factorial analysis, and methods of the structural-functional and statistical analysis. The mentioned methods are used in various combinations at different investigation phases depending on goals and the solved tasks. It promoted ensuring reliability of the carried-out analysis and validity of the conclusions drawn by authors. Results: The main results of studying of forms and methods of tax control are both the absolute measures of examination of tax authorities and relative measures presented in the article. It is impossible to give statistics of efficiency of tax monitoring. Tax monitoring in full joined into force since the beginning of 2016 and therefore statistics of efficiency are absent. Discussion: Tax monitoring is for the first time tested in practice of tax authorities of the Netherlands in 2005. Lately other countries, including the CIS countries - Ukraine, Kazakhstan, etc. also have joined introduction of horizontal monitoring. Opinions of scientists-economists in general are reduced to a positive assessment of this form of tax control that demands confirmation time. Closing statement: Emergence of a new form of tax control does not belittle value of traditional forms of control of observance of the tax law. So far, the small number of taxpayers can be passed to horizontal control because of legislative requirements for the size of the company. Parallel development of different forms of tax control will improve tax climate in the country and will allow creating conditions for creative approach in the taxation