Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.     

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.     

Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase

Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH–ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH–ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH–ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1 , nd2 , nd5 , nd6 and nd4L . Disruption of any of these genes in R . capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I-associated iron–sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs ( orf6 and orf7 ), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I-associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.     

Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli

The proton-pumping NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. In Escherichia coli the complex is made up of 13 different subunits encoded by the so-called nuo-genes. Mutants, in which each of the nuo-genes was individually disrupted by the insertion of a resistance cartridge were unable to assemble a functional complex I. Each disruption resulted in the loss of complex I-mediated activity and the failure to extract a structurally intact complex. Thus, all nuo-genes are required either for the assembly or the stability of a functional E. coli complex I. The three subunits comprising the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of several nuo-mutants as one distinct band after BN-PAGE. It is discussed that the fully assembled NADH dehydrogenase fragment represents an assembly intermediate of the E. coli complex I. A partially assembled complex I bound to the membrane was detected in the nuoK and nuoL mutants, respectively. Overproduction of the ΔNuoL variant resulted in the accumulation of two populations of a partially assembled complex in the cytoplasmic membranes. Both populations are devoid of NuoL. One population is enzymatically active, while the other is not. The inactive population is missing cluster N2 and is tightly associated with the inducible lysine decarboxylase. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

EPR signals assigned to Fe/S cluster N1c of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) derive from cluster N1a

The proton-pumping NADH:ubiquinone oxidoreductase, which is also called respiratory complex I, transfers electrons from NADH to ubiquinone via one flavin mononucleotide (FMN) and up to nine iron-sulfur clusters. A structural minimal form of complex I consisting of 14 different subunits called NuoA to NuoN (or Nqo1 to Nqo14) is found in bacteria. The isolated Escherichia coli complex I can be split into a NADH dehydrogenase fragment, a connecting fragment, and a membrane fragment. The soluble NADH dehydrogenase fragment represents the electron input part of the complex and consists of the subunits NuoE, F, and G. The FMN and four iron-sulfur clusters have been detected in this fragment by means of EPR spectroscopy. One of the EPR signals, called N1c, has spectral properties, which are not found in preparations of the complex from other organisms. Therefore, it is attributed to an additional binding motif on NuoG, which is present only in a few bacteria including E. coli. Here, we show by means of EPR spectroscopic analysis of the NADH dehydrogenase fragment containing site-directed mutations on NuoG that the EPR signals in question derived from cluster N1a on NuoE. The mutations in NuoG disturbed the assembly of the overproduced NADH dehydrogenase fragment indicating that a yet undetected cluster might be bound to the additional motif. Thus, there is no third binuclear iron-sulfur "N1c" in the E. coli complex I but an additional tetranuclear cluster that may be coined N7.     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography.

In order to isolate the mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase EC 1.6.99.3) from wheat, we developed a one-step immunoaffinity procedure using antibodies raised against the NAD9 subunit. By native electrophoresis we showed that the antibodies are able to recognize the NAD9 subunit on the complex in its native form, therefore allowing the immunoaffinity chromatography. The complex retained on the column proved to be a functional complex I, since the preparation showed NADH:duroquinone and NADH:FeK3(CN)6 reductase activities which were inhibited by rotenone. The pattern of the protein subunits (about 30) eluted from the purified complex showed a high level of similarities with complex I purified from potato and broad bean by conventional techniques. Twelve subunits were identified by cross-reactions with antibodies against heterologous complex I subunits including mitochondrial- and nuclear-encoded proteins. In order to study the genetic origin of the subunits, we purified wheat complex I after in organello labelling of mitochondrial-encoded polypeptides. We found that no other complex I subunit than those corresponding to the nine mitochondrial nad genes sequenced so far, is encoded in the mitochondria of wheat.     

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.     

Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli.

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.     

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.     

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.     

Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.     

The nuclear encoded subunits of complex I from bovine heart mitochondria

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, Iα, Iβ and Iλ, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.     

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.     

ND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed.     

Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.     

A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex.

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.     

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.     

Active/de-active transition of respiratory complex I in bacteria, fungi, and animals

Mammalian complex I (NADH:ubiquinone oxidoreductase) exists as a mixture of interconvertible active (A) and de-activated (D) forms. The A-form is capable of NADH:quinone-reductase catalysis, but not the D-form. Complex I from the bacterium Paracoccus denitrificans, by contrast, exists only in the A-form. This bacterial complex contains 32 fewer subunits than the mammalian complex. The question arises therefore if the structural complexity of complex I from higher organisms correlates with its ability to undergo the A/D transition. In the present study, it was found that complex I from the bacterium Escherichia coli and from non-vertebrate organisms (earthworm, lobster, and cricket) did not show the A/D transitions. Vertebrate organisms (carp, frog, chicken), however, underwent similar A/D transitions to those of the well-characterized bovine complex I. Further studies showed that complex I from the lower eukaryotes, Neurospora crassa and Yarrowia lipolytica, exhibited very distinct A/D transitions with much lower activation barriers compared to the bovine enzyme. The A/D transitions of complex I as they relate to structure and regulation of enzymatic activity are discussed.