The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 Å "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.

Escherichia coli complex consists of 13 subunits called NuoA to NuoN. One FMN and 7 to 8 Fe/S-clusters participate in the electron transfer reaction. The three dimensional structure of the complex from Thermus thermophilus was determined at 4.5 Å resolution [1]. However, the structure does not show electron density from the substrate quinone, thus the quinone-binding site is structurally not defined. A large cavity is located at the interface between the peripheral arm and the membrane arm which is supposed to represent the quinone-binding site. We used site-directed spin labeling (SDSL) in combination with EPR/DEER spectroscopy to localize the quinone-binding site in E. coli complex I reconstituted in lipids. For this purpose several cysteine residues were introduced at the surface of the complex. The positions were labeled with (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) containing a nitroxide radical. A decyl-ubiquinone derivative with the same label covalently linked to the end of the alkyl chain was synthesized [2]. Positions R112 B , T337 CD and A570 CD were chosen for spin labeling as the enzymatic activity of the labeled variants was not affected. The distance between the enzyme-and the substrate-bound label was measured by cw-EPR and DEER experiments. Triangulation of the distances point to a distinct position within the complex, where the alkyl chain of decyl-ubiquinone is located.NADH:quinone oxidoreductase (Complex I) is a large membrane bound enzyme complex that has evolved from smaller functional building blocks. Intermediate size enzyme complexes exist in nature, that comprise some, but not all of the protein subunits in full size complex I. The complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na + /H + antiporters, denoted MrpA and MrpD. In complex I, these subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA whereas the opposite tendency was seen for NuoL [1]. This demonstrated that although structural homologues, the MrpA-type and MrpD-type proteins have unique functional specializations. The simplest explanation for the experimental results is that MrpA and MrpD are single ion transporters that together form an antiporter. Since NuoL was previously shown to conduct Na + [2] we tentatively assigned the homologous MrpA as the Na + -channel and MrpD as the H + channel. In this work the corresponding homologous protein subunit from the smaller enzymes evolutionary related to complex I was tested in the same model system. Interestingly, the NuoL, NuoM and NuoN subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those of full size complex I, corroborating that this enzyme is indeed a bona fide complex I. The co...

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.

a b s t r a c tThe NADH:ubiquinone oxidoreductase couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. It contains a 110 Å long helix running parallel to the membrane part of the complex. Deletion of the helix resulted in a reduced H + /e À stoichiometry indicating its direct involvement in proton translocation. Here, we show that the mutation of the conserved amino acid D563 L , which is part of the horizontal helix of the Escherichia coli complex I, leads to a reduced H + /e À stoichiometry. It is discussed that this residue is involved in transferring protons to the membranous proton translocation site.

Background: Respiratory complex I accepts electrons from NADH. Results: Mutation of a single amino acid residue leads to a physiological oxidation of NADPH, however, coupled with the production of reactive oxygen species. Conclusion: The NADH-binding site of complex I evolved to discriminate NADH from NADPH and to reduce the production of reactive oxygen species. Significance: The mode of nucleotide binding determines the production of reactive oxygen species in complex I.