Complex I (NADH:ubiquinone oxidoreductase) is crucial for respiration in many aerobic organisms. In mitochondria, it oxidizes NADH from the tricarboxylic acid cycle and β-oxidation, reduces ubiquinone, and transports protons across the inner membrane, contributing to the proton-motive force. It is also a major contributor to cellular production of reactive oxygen species. The redox reaction of complex I is catalyzed in the hydrophilic domain; it comprises NADH oxidation by a flavin mononucleotide, intramolecular electron transfer along a chain of iron-sulfur clusters, and ubiquinone reduction. Redox-coupled proton translocation in the membrane domain requires long-range energy transfer through the protein complex, and the molecular mechanisms that couple the redox and proton-transfer half-reactions are currently unknown. This review evaluates extant data on the mechanisms of energy transduction and superoxide production by complex I, discusses contemporary mechanistic models, and explores how mechanistic studies may contribute to understanding the roles of complex I dysfunctions in human diseases.

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O ₂ . The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O ₂ and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD ⁺ , and the reduction potentials of the flavin and NAD ⁺ . Consequently, the ratio and concentrations of NADH and NAD ⁺ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD ⁺ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.

The biguanide metformin is widely prescribed for Type II diabetes and has anti-neoplastic activity in laboratory models. Despite evidence that inhibition of mitochondrial respiratory complex I by metformin is the primary cause of its cell-lineage-specific actions and therapeutic effects, the molecular interaction(s) between metformin and complex I remain uncharacterized. In the present paper, we describe the effects of five pharmacologically relevant biguanides on oxidative phosphorylation in mammalian mitochondria. We report that biguanides inhibit complex I by inhibiting ubiquinone reduction (but not competitively) and, independently, stimulate reactive oxygen species production by the complex I flavin. Biguanides also inhibit mitochondrial ATP synthase, and two of them inhibit only ATP hydrolysis, not synthesis. Thus we identify biguanides as a new class of complex I and ATP synthase inhibitor. By comparing biguanide effects on isolated complex I and cultured cells, we distinguish three anti-diabetic and potentially anti-neoplastic biguanides (metformin, buformin and phenformin) from two anti-malarial biguanides (cycloguanil and proguanil): the former are accumulated into mammalian mitochondria and affect oxidative phosphorylation, whereas the latter are excluded so act only on the parasite. Our mechanistic and pharmacokinetic insights are relevant to understanding and developing the role of biguanides in new and existing therapeutic applications, including cancer, diabetes and malaria.

Electron cryomicroscopy structures are provided for all core and supernumerary protein subunits of mammalian complex I, a 45-subunit enzyme that powers eukaryotic respiration. The first enzyme of the mammalian mitochondrial electron transport chain, complex I (NADH:ubiquinone oxidoreductase), is one of the largest membrane-bound enzymes in the cell. Here, Judy Hirst and colleagues report the single-particle electron cryomicroscopy structure of all 45 subunits of the bovine respiratory complex I at 4.2 Å resolution. This represents the first structure of the entire mammalian complex I, which provides insight into the structural and functional roles of the 31 'supernumerary' subunits and reveals that there are several conformationally dynamic regions that may explain how ubiquinone reduction is coupled to proton translocation. Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner mitochondrial membrane. Mammalian complex I (ref. 1) contains 45 subunits, comprising 14 core subunits that house the catalytic machinery (and are conserved from bacteria to humans) and a mammalian-specific cohort of 31 supernumerary subunits1,2. Knowledge of the structures and functions of the supernumerary subunits is fragmentary. Here we describe a 4.2-Å resolution single-particle electron cryomicroscopy structure of complex I from Bos taurus. We have located and modelled all 45 subunits, including the 31 supernumerary subunits, to provide the entire structure of the mammalian complex. Computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally dynamic regions and match biochemical descriptions of the ‘active-to-de-active’ enzyme transition that occurs during hypoxia3,4. Our structures therefore provide a foundation for understanding complex I assembly5 and the effects of mutations that cause clinically relevant complex I dysfunctions6, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.

Carbon dioxide (CO ₂ ) is a kinetically and thermodynamically stable molecule. It is easily formed by the oxidation of organic molecules, during combustion or respiration, but is difficult to reduce. The production of reduced carbon compounds from CO ₂ is an attractive proposition, because carbon-neutral energy sources could be used to generate fuel resources and sequester CO ₂ from the atmosphere. However, available methods for the electrochemical reduction of CO ₂ require excessive overpotentials (are energetically wasteful) and produce mixtures of products. Here, we show that a tungsten-containing formate dehydrogenase enzyme (FDH1) adsorbed to an electrode surface catalyzes the efficient electrochemical reduction of CO ₂ to formate. Electrocatalysis by FDH1 is thermodynamically reversible—only small overpotentials are required, and the point of zero net catalytic current defines the reduction potential. It occurs under thoroughly mild conditions, and formate is the only product. Both as a homogeneous catalyst and on the electrode, FDH1 catalyzes CO ₂ reduction with a rate more than two orders of magnitude faster than that of any known catalyst for the same reaction. Formate oxidation is more than five times faster than CO ₂ reduction. Thermodynamically, formate and hydrogen are oxidized at similar potentials, so formate is a viable energy source in its own right as well as an industrially important feedstock and a stable intermediate in the conversion of CO ₂ to methanol and methane. FDH1 demonstrates the feasibility of interconverting CO ₂ and formate electrochemically, and it is a template for the development of robust synthetic catalysts suitable for practical applications.

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Iβ, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.