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.
•Inhibition by Mg-ADP prevents ATP hydrolysis in P. denitrificans.•Deleting the C-terminus of the ε subunit did not substantially activate hydrolysis.•Deleting the ε-CTD did not compromise ATP synthesis.•P. denitrificans ATP synthase is regulated by the ε and ζ subunits and Mg-ADP.
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.
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 His
-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.
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
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.
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.
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
ATP synthase has been attributed to its unique
subunit, which is structurally analogous to the mammalian inhibitor protein IF
Here, we used homologous recombination to delete the
subunit from the
genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F
-ATPase subcomplex. ATP synthesis was not affected by loss of the
subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the
and mammalian enzymes. Therefore, deleting the
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.
Biological sex has a diverse impact on the cardiovascular system. Its influence on dilated cardiomyopathy (DCM) remains unresolved.
This study aims to investigate sex-specific differences in DCM ...presentation, natural history, and prognostic factors.
The authors conducted a prospective observational cohort study of DCM patients assessing baseline characteristics, cardiac magnetic resonance imaging, biomarkers, and genotype. The composite outcome was cardiovascular mortality or major heart failure (HF) events.
Overall, 206 females and 398 males with DCM were followed for a median of 3.9 years. At baseline, female patients had higher left ventricular ejection fraction, smaller left ventricular volumes, less prevalent mid-wall myocardial fibrosis (23% vs 42%), and lower high-sensitivity cardiac troponin I than males (all P < 0.05) with no difference in time from diagnosis, age at enrollment, N-terminal pro–B-type natriuretic peptide levels, pathogenic DCM genetic variants, myocardial fibrosis extent, or medications used for HF. Despite a more favorable profile, the risk of the primary outcome at 2 years was higher in females than males (8.6% vs 4.4%, adjusted HR: 3.14; 95% CI: 1.55-6.35; P = 0.001). Between 2 and 5 years, the effect of sex as a prognostic modifier attenuated. Age, mid-wall myocardial fibrosis, left ventricular ejection fraction, left atrial volume, N-terminal pro–B-type natriuretic peptide, high-sensitivity cardiac troponin I, left bundle branch block, and NYHA functional class were not sex-specific prognostic factors.
The authors identified a novel paradox in prognosis for females with DCM. Female DCM patients have a paradoxical early increase in major HF events despite less prevalent myocardial fibrosis and a milder phenotype at presentation. Future studies should interrogate the mechanistic basis for these sex differences.
Display omitted
An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland
in April 2022 and has now been identified in 35 countries
. Several recent studies have suggested an ...association with human adenovirus with this outbreak, a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus 2 (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing, PCR with reverse transcription, serology and in situ hybridization, we detected recent infection with AAV2 in plasma and liver samples in 26 out of 32 (81%) cases of hepatitis compared with 5 out of 74 (7%) of samples from unaffected individuals. Furthermore, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsy samples. In keeping with a CD4
T-cell-mediated immune pathology, the human leukocyte antigen (HLA) class II HLA-DRB1*04:01 allele was identified in 25 out of 27 cases (93%) compared with a background frequency of 10 out of 64 (16%; P = 5.49 × 10
). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a co-infection with human adenovirus that is usually required as a 'helper virus' to support AAV2 replication) and disease susceptibility related to HLA class II status.
Cardiac dysfunction and myocarditis have emerged as serious complications of multisystem inflammatory syndrome in children (MIS-C) and vaccines against SARS-CoV-2. Understanding the role of ...autoantibodies in these conditions is essential for guiding MIS-C management and vaccination strategies in children.
To investigate the presence of anticardiac autoantibodies in MIS-C or COVID-19 vaccine-induced myocarditis.
This diagnostic study included children with acute MIS-C or acute vaccine myocarditis, adults with myocarditis or inflammatory cardiomyopathy, healthy children prior to the COVID-19 pandemic, and healthy COVID-19 vaccinated adults. Participants were recruited into research studies in the US, United Kingdom, and Austria starting January 2021. Immunoglobulin G (IgG), IgM, and IgA anticardiac autoantibodies were identified with immunofluorescence staining of left ventricular myocardial tissue from 2 human donors treated with sera from patients and controls. Secondary antibodies were fluorescein isothiocyanate-conjugated antihuman IgG, IgM, and IgA. Images were taken for detection of specific IgG, IgM, and IgA deposits and measurement of fluorescein isothiocyanate fluorescence intensity. Data were analyzed through March 10, 2023.
IgG, IgM and IgA antibody binding to cardiac tissue.
By cohort, there were a total of 10 children with MIS-C (median IQR age, 10 13-14 years; 6 male), 10 with vaccine myocarditis (median age, 15 14-16 years; 10 male), 8 adults with myocarditis or inflammatory cardiomyopathy (median age, 55 46-63 years; 6 male), 10 healthy pediatric controls (median age, 8 13-14 years; 5 male), and 10 healthy vaccinated adults (all older than 21 years, 5 male). No antibody binding above background was observed in human cardiac tissue treated with sera from pediatric patients with MIS-C or vaccine myocarditis. One of the 8 adult patients with myocarditis or cardiomyopathy had positive IgG staining with raised fluorescence intensity (median IQR intensity, 11 060 10 223-11 858 AU). There were no significant differences in median fluorescence intensity in all other patient cohorts compared with controls for IgG (MIS-C, 6033 5834-6756 AU; vaccine myocarditis, 6392 5710-6836 AU; adult myocarditis or inflammatory cardiomyopathy, 5688 5277-5990 AU; healthy pediatric controls, 6235 5924-6708 AU; healthy vaccinated adults, 7000 6423-7739 AU), IgM (MIS-C, 3354 3110-4043 AU; vaccine myocarditis, 3843 3288-4748 AU; healthy pediatric controls, 3436 3313-4237 AU; healthy vaccinated adults, 3543 2997-4607 AU) and IgA (MIS-C, 3559 2788-4466 AU; vaccine myocarditis, 4389 2393-4780 AU; healthy pediatric controls, 3436 2425-4077 AU; healthy vaccinated adults, 4561 3164-6309 AU).
This etiological diagnostic study found no evidence of antibodies from MIS-C and COVID-19 vaccine myocarditis serum binding cardiac tissue, suggesting that the cardiac pathology in both conditions is unlikely to be driven by direct anticardiac antibody-mediated mechanisms.
PDF
