Hannah R. Bridges

Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria

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.

Mossbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized.

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron–sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use Mössbauer spectroscopy on 57Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.

The Subunit Composition of Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) From Pichia pastoris

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

Investigating the function of [2Fe–2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential

NADH:quinone oxidoreductase (complex I) couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. All complexes I contain a flavin to oxidize NADH, seven iron–sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (N1a) on the opposite side of the flavin. The role of cluster N1a is unknown, but Escherichia coli complex I has an unusually high-potential cluster N1a and its reduced flavin produces H2O2, not superoxide, suggesting that cluster N1a may affect reactive oxygen species production. In the present study, we combine protein film voltammetry with mutagenesis in overproduced N1a-binding subunits to identify two residues that switch N1a between its high- (E. coli, valine and asparagine) and low- (Bos taurus and Yarrowia lipolytica, proline and methionine) potential forms. The mutations were incorporated into E. coli complex I: cluster N1a could no longer be reduced by NADH, but H2O2 and superoxide production were unaffected. The reverse mutations (that increase the potential by ~0.16 V) were incorporated into Y. lipolytica complex I, but N1a was still not reduced by NADH. We conclude that cluster N1a does not affect reactive oxygen species production by the complex I flavin; it is probably required for enzyme assembly or stability.

The mitochondrial-encoded subunits of respiratory complex I (NADH:ubiquinone oxidoreductase): identifying residues important in mechanism and disease

Complex I (NADH:ubiquinone oxidoreductase) is crucial to respiration in many aerobic organisms. The hydrophilic domain of complex I, containing nine or more redox cofactors, and comprising seven conserved core subunits, protrudes into the mitochondrial matrix or bacterial cytoplasm. The α-helical membrane-bound hydrophobic domain contains a further seven core subunits that are mitochondrial-encoded in eukaryotes and named the ND subunits (ND1-ND6 and ND4L). Complex I couples the oxidation of NADH in the hydrophilic domain to ubiquinone reduction and proton translocation in the hydrophobic domain. Although the mechanisms of NADH oxidation and intramolecular electron transfer are increasingly well understood, the mechanisms of ubiquinone reduction and proton translocation remain only poorly defined. Recently, an α-helical model of the hydrophobic domain of bacterial complex I [Efremov, Baradaran and Sazanov (2010) Nature 465, 441-447] revealed how the 63 transmembrane helices of the seven core subunits are arranged, and thus laid a foundation for the interpretation of functional data and the formulation of mechanistic proposals. In the present paper, we aim to correlate information from sequence analyses, site-directed mutagenesis studies and mutations that have been linked to human diseases, with information from the recent structural model. Thus we aim to identify and discuss residues in the ND subunits of mammalian complex I which are important in catalysis and for maintaining the enzymes structural and functional integrity.

The respiratory complexes I from the mitochondria of two Pichia species

NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinsons disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. In the present study, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple medium, as yeast-like, spheroidal cells. Both Pichia enzymes catalyse inhibitor-sensitive NADH:ubiquinone oxidoreduction, display EPR spectra which match closely to those from other eukaryotic complexes I, and show patterns characteristic of complex I in SDS/PAGE analysis. Mass spectrometry was used to identify several canonical complex I subunits. Purified P. pastoris complex I has a particularly high specific activity, and incorporating it into liposomes demonstrates that NADH oxidation is coupled to the generation of a protonmotive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as high as that of the Bos taurus enzyme. Our results both resolve previous disagreement about whether Pichia species encode complex I, furthering understanding of the evolution of complex I within dikarya, and they provide two new, robust and highly active model systems for study of the structure and catalytic mechanism of eukaryotic complexes I.

Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase

BackgroundThe biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known.ResultsHere, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments.ConclusionsBiguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies.

Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system

Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction.

A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase

Abstract Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone‐10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self‐assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady‐state catalysis. The system was used to measure a fully‐defined K M value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.