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.

Redox components and structure of the respiratory NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase is the first complex in the respiratory chains of many purple bacteria and of mitochondria of most eucaryotes. The bacterial complex consists of 14 different subunits. The mitochondrial complex contains at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. We analysed electron micrographs of isolated and negatively stained complex I particles from Escherichia coli and Neurospora crassa and obtained three-dimensional models of both complexes at medium resolution. Both have the same L-shaped overall structure with a peripheral arm protruding into the aqueous phase and a membrane arm extending into the membrane. The two arms of the bacterial complex are only slightly shorter than those of the mitochondrial complex although the protein mass of the former is only half of that of the latter. The presence of a novel redox group in the membrane arm of the complex is discussed. This group has been detected in the N. crassa complex by means of UV-visible spectroscopy. After reduction with an excess of NADH and reoxidation by the lactate dehydrogenase reaction, a reduced-minus-oxidized difference spectrum was obtained that cannot be attributed to the known cofactors flavin mononucleotide (FMN) and the FeS clusters N1, N2, N3 and N4. Due to its positive midpoint potential the novel group is believed to transfer electrons from the FeS clusters to ubiquinone. Its role in proton translocation is discussed.

The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for energy consuming processes like the synthesis of ATP. Homologues of this complex exist in bacteria, archaea, in mitochondria of eukaryotes and in chloroplasts of plants. The bacterial and mitochondrial complexes function as NADH dehydrogenase, while the archaeal complex works as F420H2 dehydrogenase. The electron donor of the cyanobacterial and plastidal complex is not yet known. Despite the different electron input sites, 11 polypeptides constitute the structural framework for proton translocation and quinone binding in the complex of all three domains of life. Six of them are also present in a family of membrane-bound multisubunit [NiFe] hydrogenases. It is discussed that they build a module for electron transfer coupled to proton translocation.

Identification of two tetranuclear FeS clusters on the ferredoxin-type subunit of NADH:ubiquinone oxidoreductase (complex I).

The proton-translocating NADH:ubiquinone oxidoreductase of respiratory chains (complex I) contains one flavin mononucleotide and five EPR-detectable iron-sulfur clusters as redox groups. Because of the number of conserved motifs typical for binding iron-sulfur clusters and the high content of iron and acid-labile sulfide of complex I preparations, it is predicted that complex I contains additional clusters which have not yet been detected by EPR spectroscopy. To search for such clusters, we used a combination of UV/vis and EPR spectroscopy to study complex I from Neurospora crassa and Escherichia coli adjusted to distinct redox states. We detected a UV/vis redox difference spectrum characterized by negative absorbances at 325 and 425 nm that could not be assigned to the known redox groups. Redox titration was used to determine the pH-independent midpoint potential to be -270 mV, being associated with the transfer of two electrons. Comparison with UV/vis difference spectra obtained from complex I fragments and related enzymes showed that this group is localized on subunit Nuo21.3c of the N. crassa or NuoI of the E. coli complex I, respectively. This subunit (the bovine TYKY) belongs to a family of 8Fe-ferredoxins which contain two tetranuclear iron-sulfur clusters as redox groups. We detected EPR signals in a fragment of complex I which we attribute to the novel FeS clusters of complex I.

Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.

FT-IR spectroscopic characterization of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli: oxidation of FeS cluster N2 is coupled with the protonation of an aspartate or glutamate side chain.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) clusters participate in the redox reaction. So far, complex I has been described mainly by means of EPR- and UV-vis spectroscopy. Here, we report for the first time an infrared spectroscopic characterization of complex I. Electrochemically induced FT-IR difference spectra of complex I from Escherichia coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in both preparations were derived from a comparison using model compounds and turned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induce strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transition of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorganization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.