Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different subunits. In addition to the homologues of these subunits, the mitochondrial complex contains approximately 31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli mutants, in which the nuo-genes coding the subunits of complex I were individually disrupted by an insertion of a resistance cartridge to determine whether they are required for the assembly of a functional complex I. No complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron-sulfur cluster N2 was detected in one mutant.     

Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits

NADH:ubiquinone reductase, the respiratory chain complex I of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one FMN, probably one internal ubiquinone and at least four iron-sulphur clusters. We are studying the assembly of the enzyme in Neurospora crassa. The flux of radioactivity in cells that were pulse-labelled with [35S]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. Labelled polypeptides were observed to accumulate transiently in a Mr 350,000 intermediate complex. This complex contains all mitochondrially encoded subunits of the enzyme as well as subunits encoded in the nucleus that have no homologous counterparts in a small, merely nuclear-encoded form of the NADH:ubiquinone reductase made by Neurospora crassa cells poisoned with chloramphenicol. With regard to their subunit compositions, the assembly intermediate and small NADH:ubiquinone reductase complement each other almost perfectly to give the subunit composition of the large complex I. These results suggest that two pathways exist in the assembly of complex I that independently lead to the preassembly of two major parts, which subsequently join to form the complex. One preassembled part is related to the small form of NADH:ubiquinone reductase and contributes most of the nuclear-encoded subunits, FMN, three iron-sulphur clusters and the site for the internal ubiquinone. The other part is the assembly intermediate and contributes all mitochondrially encoded subunits, one iron-sulphur cluster and the catalytic site for the substrate ubiquinone. We discuss the results with regard to the evolution of the electron pathway through complex I.     

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.     

Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.     

Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

Iron-sulfur cluster N7 of the NADH:ubiquinone oxidoreductase (complex I) is essential for stability but not involved in electron transfer

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN. It catalyzes the electron transfer from NADH to ubiquinone by a chain of redox groups consisting of one FMN and seven iron-sulfur clusters. The function of the additional, nonconserved cluster N7 located on NuoG is not known. It has been speculated that it is not involved in electron transfer, due to its distance of more than 20 A from the electron transfer chain. Dithionite-reduced minus NADH-reduced EPR difference spectra of complex I and of a soluble fragment containing NuoG revealed for the first time the EPR spectrum of N7 in the complex. Individual mutation of the cysteines ligating this cluster to alanine led to a decreased amount of complex I in the membrane without affecting the electron transfer activity. Sucrose gradient centrifugation revealed that the complex from the C230A and C233A mutants decayed in detergent solution while the C237A and C265A mutant complex was stable. Cluster N7 was detectable in the latter mutants but with shifted g-values, indicating a different ligation of N7. Thus, N7 is essential for the stability of the complex but is not involved in electron transfer.     

Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

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 enzyme's structural and functional integrity.     

Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays

Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the respiratory electron transport chain. Homologs of this complex exist in bacteria, mitochondria and chloroplasts. The minimal complex I from mitochondria and bacteria contains 14 different subunits grouped into three modules: membrane, connecting, and soluble subcomplexes. The complex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts from higher plants contains genes for two out of three modules: the membrane and connecting subcomplexes. However, there is not much information about the existence of the soluble subcomplex (which is the electron input device in bacterial complex I) in the composition of the Ndh complex. Furthermore, there are contrasting reports regarding the subunit composition of the Ndh complex and its molecular mass. By using blue native (BN)/PAGE and Tricine/PAGE or colorless-native (CN)/PAGE, BN/PAGE and Tricine/PAGE, combined with mass spectrometry, we attempted to obtain more information about the plastidal Ndh complex from maize (Zea mays). Using antibodies, we detected the expression of a new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloroplasts and in ethioplasts (ET). We determined the molecular mass of the Ndh complex (550 kDa) and observed that it splits into a 300 kDa membrane subcomplex (containing NdhE) and a 250 kDa subcomplex (containing NdhH, -J and -K). The Ndh complex forms dimers at 1000-1100 kDa in both MS and BS chloroplasts. Native/PAGE of the MS and BS chloroplasts allowed us to determine that the Ndh complex contains at least 14 different subunits. The native gel electrophoresis, western blotting and mass spectrometry allowed us to identify five of the Ndh subunits. We also provide a method that allows the purification of large amounts of Ndh complex for further structural, as well as functional studies.     

Characterization of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption.

NADH:ubiquinone oxidoreductase, the respiratory chain complex I of mitochondria, is an assembly of some 25 nuclear-encoded and 7 mitochondrially encoded subunits. The complex has an overall L-shaped structure formed by a peripheral arm and an elongated membrane arm. The peripheral arm containing one FMN and at least three iron-sulphur clusters constitutes the NADH dehydrogenase segment of the electron pathway. The membrane arm with at least one iron-sulphur cluster constitutes the ubiquinone reducing segment. We are studying the assembly of the complex in Neurospora crassa. By disrupting the gene of a nuclear-encoded subunit of the membrane arm a mutant was generated that cannot form complex I. The mutant rather pre-assembles the peripheral arm with all redox groups and the ability to catalyse NADH oxidation by artificial electron acceptors. The final assembly of the membrane arm is blocked in the mutant leading to accumulation of complementary assembly intermediates. One intermediate is associated with a protein that is not present in the fully assembled complex I. The results demonstrate that the two arms of complex I are assembled independently on separate pathways, and gave a first insight into the assembly pathway of the membrane arm. It is also shown for the first time that the obligate aerobic fungus N. crassa can grow and respire without an intact complex I. Gene replacement in this fungus is therefore a tool for investigation of this complex.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The long-known reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430–1436) disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90?Å long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature.     

Challenges in elucidating structure and mechanism of proton pumping NADH:ubiquinone oxidoreductase (complex I)

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the most complicated and least understood enzyme of the respiratory chain. All redox prosthetic groups reside in the peripheral arm of the L-shaped structure. The NADH oxidation domain harbouring the FMN cofactor is connected via a chain of iron–sulfur clusters to the ubiquinone reduction site that is located in a large pocket formed by the PSST- and 49-kDa subunits of complex I. An access path for ubiquinone and different partially overlapping inhibitor binding regions were defined within this pocket by site directed mutagenesis. A combination of biochemical and single particle analysis studies suggests that the ubiquinone reduction site is located well above the membrane domain. Therefore, direct coupling mechanisms seem unlikely and the redox energy must be converted into a conformational change that drives proton pumping across the membrane arm. It is not known which of the subunits and how many are involved in proton translocation. Complex I is a major source of reactive oxygen species (ROS) that are predominantly formed by electron transfer from FMNH₂. Mitochondrial complex I can cycle between active and deactive forms that can be distinguished by the reactivity towards divalent cations and thiol-reactive agents. The physiological role of this phenomenon is yet unclear but it could contribute to the regulation of complex I activity in-vivo.     

Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.     

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida)

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.     

The oxidized subunit B8 from human complex I adopts a thioredoxin fold

Subunit B8 from ubiquinone oxidoreductase (complex I) (CI-B8) is one of several nuclear-encoded supernumerary subunits that are not present in bacterial complex I. Its solution structure shows a thioredoxin fold with highest similarities to the human thioredoxin mutant C73S and thioredoxin 2 from Anabeana sp. Interestingly, these proteins contain active sites in the same area, where the disulfide bond of oxidized CI-B8 is located. The redox potential of this disulfide bond is -251.6 mV, comparing well to that of disulfides in other thioredoxin-like proteins. Analysis of the structure reveals a surface area that is exclusively composed of highly conserved residues and thus most likely a subunit interaction site within complex I.