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

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

Key role of quinone in the mechanism of respiratory complex I

Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance