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The first component of the mitochondrial electron transport chain is respiratory complex I. Several high-resolution structures of complex I from different species have been resolved. However, despite these significant achievements, the mechanism of redox-coupled proton pumping remains elusive. Here, we combined atomistic docking, molecular dynamics simulations, and site-directed mutagenesis on respiratory complex I from Yarrowia lipolytica to identify a quinone (Q)-binding site on its surface near the horizontal amphipathic helices of ND1 and NDUFS7 subunits. The surface-bound Q makes stable interactions with conserved charged and polar residues, including the highly conserved Arg72 from the NDUFS7 subunit. The binding and dynamics of a Q molecule at the surface-binding site raise interesting possibilities about the mechanism of complex I, which are discussed.

Description, High-resolution structure and molecular simulations unravel the inner workings of a redox-driven proton pump., Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1-MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined, but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1-Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ~100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4-Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamic simulations, we define details of the proton translocation pathways and offer insights into the redox-coupled proton pumping mechanism of complex I.

SummaryMitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ∼100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.

Respiratory complex I catalyses the reduction of ubiquinone (Q) from NADH coupled to proton pumping across the inner membrane of mitochondria. The electrical charging of the inner mitochondrial membrane drives the synthesis of ATP, which is used to power biochemical reactions of the cell. The recent surge in structural data on complex I from bacteria and mitochondria have contributed to significant understanding of its molecular architecture. However, despite these accomplishments, the role of various subdomains in redox-coupled proton pumping remains entirely unclear. In this work, we have mutated conserved residues in the loop of the PSST subunit that faces the similar to 30 angstrom long unique Q-binding tunnel of respiratory complex I. The data show a drastic decrease in Q reductase activity upon mutating several residues despite full assembly of the complex. In-silico modeling and multiple microsecond long molecular dynamics simulations of wild-type and enzyme variants with exchanges of conserved arginine residues revealed remarkable ejection of the bound Q from the site near terminal electron donor N2. Based on experiments and long-time scale molecular simulations, we identify microscopic elements that dynamically control the diffusion of Q and are central to redox-coupled proton pumping in respiratory complex I.

Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48 Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system., This work was financially supported by grants from the Deutsche Forschungsgemeinschaft - DFG (GRK1673 to H.K.; Ku961/11-1 to H.K.; SFB740 to P.S.; SFB1078 to P.S.) and DFG - Cluster of Excellence ‘Unifying Concepts in Catalysis’ (Research Field D3/E3-1 to J.K. and P.S.).

Secreted LOX from Pseudomonas aeruginosa (PA-LOX) has previously been identified as arachidonic acid 15S-lipoxygenating enzyme. Here we report that the substitution of Ala420Gly in PA-LOX leads to an enzyme variant with pronounced dual specificity favoring arachidonic acid 11R-oxygenation. When compared with other LOX-isoforms the molecular oxygen affinity of wild-type PA-LOX is 1–2 orders of magnitude lower (Km O2 of 0.4 mM) but Ala420Gly exchange improved the molecular oxygen affinity (Km O2 of 0.2 mM). Experiments with stereo-specifically deuterated linoleic acid indicated that the formation of both 13S- and 9R-HpODE involves abstraction of the proS-hydrogen from C11 of the fatty acid backbone. To explore the structural basis for the observed functional changes (altered specificity, improved molecular oxygen affinity) we solved the crystal structure of the Ala420Gly mutant of PA-LOX at 1.8 A resolution and compared it with the wild-type enzyme. Modeling of fatty acid alignment at the catalytic center suggested that in the wild-type enzyme dioxygen is directed to C15 of arachidonic acid by a protein tunnel, which interconnects the catalytic center with the protein surface. Ala420Gly exchange redirects intra-enzyme O2 diffusion by bifurcating this tunnel so that C11 of arachidonic acid also becomes accessible for O2 insertion.

An unusual behavior of the periplasmic aldehyde oxidoreductase (PaoABC) from Escherichia coli has been observed from electrochemical investigations of the enzyme catalyzed oxidation of aromatic aldehydes with different mediators under different conditions of ionic strength. The enzyme has similarity to other molybdoenzymes of the xanthine oxidase family, but the catalytic behavior turned out to be very different. Under steady state conditions the turnover of PaoABC is maximal at pH 4 for the negatively charged ferricyanide and at pH 9 for a positively charged osmium complex. Stopped-flow kinetic measurements of the catalytic half reaction showed that oxidation of benzaldehyde proceeds also above pH 7. Thus, benzaldehyde oxidation can proceed under acidic and basic conditions using this enzyme, a property which has not been described before for molybdenum hydroxylases. It is also suggested that the electron transfer with artificial electron acceptors and PaoABC can proceed at different protein sites and depends on the nature of the electron acceptor in addition to the ionic strength. Keywords: Electron transfer, Multi-cofactor enzymes, Molybdoenzymes, Aldehyde oxidoreductase