Membrane-domain mutations in respiratory complex I impede catalysis but do not uncouple proton pumping from ubiquinone reduction.

Respiratory complex I [NADH:ubiquinone (UQ) oxidoreductase] captures the free energy released from NADH oxidation and UQ reduction to pump four protons across an energy-transducing membrane and power ATP synthesis. Mechanisms for long-range energy coupling in complex I have been proposed from structural data but not yet evaluated by robust biophysical and biochemical analyses. Here, we use the powerful bacterial model system Paracoccus denitrificans to investigate 14 mutations of key residues in the membrane-domain Nqo13/ND4 subunit, defining the rates and reversibility of catalysis and the number of protons pumped per NADH oxidized. We reveal new insights into the roles of highly conserved charged residues in lateral energy transduction, confirm the purely structural role of the Nqo12/ND5 transverse helix, and evaluate a proposed hydrated channel for proton uptake. Importantly, even when catalysis is compromised the enzyme remains strictly coupled (four protons are pumped per NADH oxidized), providing no evidence for escape cycles that circumvent blocked proton-pumping steps.

Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I.

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His6-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans.

F1FO-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Deleting the IF1-like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis.

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF1 Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F1-ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.