Stefan Steimle

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.

Asp563 of the horizontal helix of subunit NuoL is involved in proton translocation by the respiratory complex I.

The 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− stoichiometry indicating its direct involvement in proton translocation. Here, we show that the mutation of the conserved amino acid D563L, which is part of the horizontal helix of the Escherichia coli complex I, leads to a reduced H+/e− stoichiometry. It is discussed that this residue is involved in transferring protons to the membranous proton translocation site.

Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

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. The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.

Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston‐like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine‐scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin‐probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement.

Involvement of Acidic Amino Acid Residues in Zn2+ Binding to Respiratory Complex I

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn2+ ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn2+inhibition of the proton‐pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm−1) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn2+ binding. In variant D563NL, which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn2+ binding is changed. Our data support a role for this residue in proton translocation.