Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.

Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.

Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.

Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme.

Respiratory Complex I (NADH:ubiquinone oxidoreductase) is composed of 45 subunits, seven mitochondrially-encoded and 38 imported. Mutations in the nuclearly-encoded subunits have been regularly discovered in humans in recent years, and many lead to cardiomyopathy, Leigh Syndrome, and early death. From the literature, we have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. Many of these mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that they would disrupt assembly of Complex I. The mutations were constructed in the homologous E. coli genes, nuoG, nuoCD, nuoI and nuoF, respectively, and expressed from a plasmid containing all Complex I genes. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity measured, which indicated a range of reduced activity. Some mutants were also analyzed using recently developed assays of assembly, time-delayed expression, and co-immunoprecipitation, which showed that assembly was disrupted. With compound heterozygotes, we determined which mutation was more deleterious. Construction of alanine mutations allowed us to distinguish between phenotypes that were caused by loss of the original amino acid or introduction of the mutant residue.

Human clinical mutations in mitochondrially encoded subunits of Complex I can be successfully modeled in E. coli.

Respiratory Complex I is the site of a large fraction of the mutations that appear to cause mitochondrial disease. Seven of its subunits are mitochondrially encoded, and therefore, such mutants are particularly difficult to construct in cell-culture model systems. We have selected 13 human clinical mutations found in ND2, ND3, ND4, ND4L, ND5 and ND6 that are generally found at subunit interfaces, and not in critical residues. These mutations have been modeled in E. coli subunits of Complex I, nuoN, nuoA, nuoM, nuoK, nuoL, and nuoJ, respectively. All mutants were expressed from a plasmid encoding the entire nuo operon, and membrane vesicles were analyzed for deamino-NADH oxidase activity, and proton translocation activity. ND5 mutants were also analyzed using a time-delayed expression system, recently described by this lab. Other mutants were analyzed for the ability to associate in subcomplexes, after expression of subsets of the genes. For most mutants there was a positive correlation between those that were previously determined to be pathogenic, or likely to be pathogenic, and those that we found with compromised Complex I activity or subunit interactions in E. coli. In conclusion, this approach provides another way to explore the deleterious effects of human mitochondrial mutations, and it can contribute to molecular understanding of such mutations.

Analysis of Human Clinical Mutations of Mitochondrial ND1 in a Bacterial Model System for Complex I.

The most common causes of mitochondrial dysfunction and disease include mutations in subunits and assembly factors of Complex I. Numerous mutations in the mitochondrial gene ND1 have been identified in humans. Currently, a bacterial model system provides the only method for rapid construction and analysis of mutations in homologs of human ND1. In this report, we have identified nine mutations in human ND1 that are reported to be pathogenic and are located at subunit interfaces. Our hypothesis was that these mutations would disrupt Complex I assembly. Seventeen mutations were constructed in the homologous nuoH gene in an E. coli model system. In addition to the clinical mutations, alanine substitutions were constructed in order to distinguish between a deleterious effect from the introduction of the mutant residue and the loss of the original residue. The mutations were moved to an expression vector containing all thirteen genes of the E. coli nuo operon coding for Complex I. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity and proton translocation were measured. Samples were also tested for assembly by native gel electrophoresis and for expression of NuoH by immunoblotting. A range of outcomes was observed: Mutations at four of the sites allow normal assembly with moderate activity (50−76% of wild type). Mutations at the other sites disrupt assembly and/or activity, and in some cases the outcomes depend upon the amino acid introduced. In general, the outcomes are consistent with the proposed pathogenicity in humans.

Subunits E-F-G of E. coli Complex I can form an active complex when expressed alone, but in time-delayed assembly co-expression of B-CD-E-F-G is optimal.

Respiratory Complex I from E. coli is a proto-type of the mitochondrial enzyme, consisting of a 6-subunit peripheral arm (B-CD-E-F-G-I) and a 7-subunit membrane arm. When subunits E-F-G (N-module), were expressed alone they formed an active complex as determined by co-immunoprecipitation and native gel electrophoresis. When co-expressed with subunits B and CD, only a complex of E-F-G was found. When these five subunits were co-expressed with subunit I and two membrane subunits, A and H, a complex of B-CD-E-F-G-I was membrane-bound, constituting the N- and Q-modules. Assembly of Complex I was also followed by splitting the genes between two plasmids, in three different groupings, and expressing them simultaneously, or with time-delay of expression from one plasmid. When the B-CD-E-F-G genes were co-expressed after a time-delay, assembly was over 90 % of that when the whole operon was expressed together. In summary, E-F-G was the only soluble subcomplex detected in these studies, but assembly was not optimal when these subunits were expressed either first or last. Co-expression of subunits B and CD with E-F-G provided a higher level of assembly, indicating that integrated assembly of N- and Q-modules provides a more efficient pathway.

Analysis of the assembly pathway for membrane subunits of Complex I reveals that subunit L (ND5) can assemble last in E. coli.

Respiratory Complex I, a multi-subunit, membrane-bound enzyme, oxidizes NADH in the electron transport chains of mammalian mitochondria, and many bacterial species. We have examined in vivo assembly of the membrane subunits of Complex I from E. coli. Complexes of J-K, L-M, M-N, and J-K-L-M-N were observed by both native gel electrophoresis and co-immunoprecipitation, when subsets of the genes were expressed. Subunit L (ND5 in humans), the most distal membrane subunit, with an unusual extended C-terminal segment, did not join with M-N, and but could join with J-K-M-N. When the genes were split between two plasmids, with L, M, and N subunits expressed in various combinations from one plasmid, the resulting enzyme activity in membrane vesicles dropped to 19-60% relative to expression from the whole operon encoded on one plasmid. When L was expressed after a time-delay, rather than simultaneously, the activity increased from 28% to 100%, indicating that it can efficiently join a preformed complex lacking L. In contrast, when larger groups of membrane subunits were expressed last, LMN or JKLMN, assembly was much less efficient. The two-plasmid expression system was used to re-analyze C-terminal mutations in subunit K (ND4L), which occur near the overlapping nuoK and L genes. These mutations were found to disrupt assembly, indicating the importance of the junction of L, N and K subunits. The results highlight the temporal and spatial aspects of gene expression that allow efficient assembly of the membrane subunits of Complex I.

A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase.

Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.

Transmembrane topology of subunit N of complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli.

The transmembrane topology of subunit N from E. coli Complex I has been investigated. Chemical labeling of mono-substituted cysteine mutants was carried out in inverted membrane vesicles, and in whole cells, using 3-N-maleimidyl-propionyl biocytin (MPB). The results support a model of 14 transmembrane spans with both termini in the periplasm, and are consistent with the models of subunits L, M and N from the crystal structure of the membrane arm of the E. coli Complex I (Efremov et al. (2010) Nature 465, 441-445). In particular, the results do not support an unusual cytoplasmic localization of two likely transmembrane regions, as proposed in previous studies (Mathiesen and Hägerhäll (2002) Biochim Biophys Acta 1556, 121-132; Torres-Bacete, et al. (2009) J Biol Chem 284, 33062-33069).

Analysis of Human Mutations in the Supernumerary Subunits of Complex I.

Complex I is the largest member of the electron transport chain in human mitochondria. It comprises 45 subunits and requires at least 15 assembly factors. The subunits can be divided into 14 "core" subunits that carry out oxidation-reduction reactions and proton translocation, as well as 31 additional supernumerary (or accessory) subunits whose functions are less well known. Diminished levels of complex I activity are seen in many mitochondrial disease states. This review seeks to tabulate mutations in the supernumerary subunits of humans that appear to cause disease. Mutations in 20 of the supernumerary subunits have been identified. The mutations were analyzed in light of the tertiary and quaternary structure of human complex I (PDB id = 5xtd). Mutations were found that might disrupt the folding of that subunit or that would weaken binding to another subunit. In some cases, it appeared that no protein was made or, at least, could not be detected. A very common outcome is the lack of assembly of complex I when supernumerary subunits are mutated or missing. We suggest that poor assembly is the result of disrupting the large network of subunit interactions that the supernumerary subunits typically engage in.

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase.

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N'-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F(1)-F(o) interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to >10 microns, as well as ATP production by this chain.

Construction of a deletion strain and expression vector for the Escherichia coli NADH:ubiquinone oxidoreductase (Complex I).

Complex I of Escherichia coli is encoded by 13 consecutive genes, called the nuo operon. A chromosomal deletion of all nuo genes has been achieved by homologous recombination. A vector that encodes all of the nuo genes has been constructed, and it expresses a functional enzyme.

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase.

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase.

His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.

Direct transfer of NADH from malate dehydrogenase to complex I in Escherichia coli.

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH:ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate + NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate + NAD was estimated to be 1 microM, while the rate of proton translocation due to Complex I was consistent with a somewhat higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.

Interactions between subunits a and b in the rotary ATP synthase as determined by cross-linking.

The interaction of the membrane traversing stator subunits a and b of the rotary ATP synthase was probed by substitution of a single Cys into each subunit with subsequent Cu(2+) catalyzed cross-linking. Extensive interaction between the transmembrane (TM) region of one b subunit and TM2 of subunit a was indicated by cross-linking with 6 Cys pairs introduced into these regions. Additional disulfide cross-linking was observed between the N-terminus of subunit b and the periplasmic loop connecting TM4 and TM5 of subunit a. Finally, benzophenone-4-maleimide derivatized Cys in the 2-3 periplasmic loop of subunit a were shown to cross-link with the periplasmic N-terminal region of subunit b. These experiments help to define the juxtaposition of subunits b and a in the ATP synthase.

Theaflavins inhibit the ATP synthase and the respiratory chain without increasing superoxide production.

Four dietary polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (TF3), have been isolated from black tea, and their effects on oxidative phosphorylation and superoxide production in a model system (Escherichia coli) have been examined. The esterified theaflavins were all potent inhibitors of the membrane-bound adenosine triphosphate (ATP) synthase, inhibiting at least 90% of the activity, with IC(50) values in the range of 10-20 µM. ATP-driven proton translocation was inhibited in a similar fashion, as was the purified F(1)-ATPase, indicating that the primary site of inhibition was in the F(1) sector. Computer modeling studies supported this interpretation. All four theaflavins were also inhibitory towards the electron transport chain, whether through complex I (NDH-1) or the alternative NADH dehydrogenase (NDH-2). Inhibition of NDH-1 by TF3 appeared to be competitive with respect to NADH, and this was supported by computer modeling studies. Rates of superoxide production during NADH oxidation by each dehydrogenase were measured. Superoxide production was completely eliminated in the presence of about 15 µM TF3, suggesting that inhibition of the respiratory chain by theaflavins does not contribute to superoxide production.