scholarly journals Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes

2018 ◽  
Vol 293 (43) ◽  
pp. 16687-16696 ◽  
Author(s):  
Chang-Hao Wu ◽  
Gerrit J. Schut ◽  
Farris L. Poole ◽  
Dominik K. Haja ◽  
Michael W. W. Adams
Keyword(s):  
Complex I ◽  
Pyrococcus Furiosus ◽  
Atp Synthesis ◽  
Genetically Engineered ◽  
Hydrogen Gas ◽  
Hyperthermophilic Archaea ◽  
Cysteine Residues ◽  
Sulfane Sulfur ◽  
Respiratory Complexes ◽  
Membrane Bound

Hyperthermophilic archaea contain a hydrogen gas–evolving,respiratory membrane–bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H2S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable Km (∼490 μm) and Vmax values (12 μmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd (Eo′ = −480 mV) to reduce sulfane sulfur (Eo′ −260 mV) and cleave organic (RSnR, n ≥ 3) and anionic polysulfides (Sn2−, n ≥ 4) but that it does not produce H2S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

scholarly journals pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus

2021 ◽  
Vol 12 ◽  
Author(s):  
Dominik K. Haja ◽  
Michael W. W. Adams
Keyword(s):  
Pyrococcus Furiosus ◽  
Gas Production ◽  
Hydrogen Gas ◽  
Sodium Ion ◽  
Redox Activity ◽  
Deletion Strain ◽  
Multiple Resistance ◽  
Ph Homeostasis ◽  
Membrane Bound ◽  
Ion Pumping

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.

scholarly journals An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

2020 ◽  
Author(s):  
Daniel Missailidis ◽  
Sarah Annesley ◽  
Claire Allan ◽  
Oana Sanislav ◽  
Brett Lidbury ◽  
...  
Keyword(s):  
Steady State ◽  
Fatty Acid ◽  
Mitochondrial Function ◽  
Complex I ◽  
Atp Synthesis ◽  
Beta Oxidation ◽  
Respiratory Capacity ◽  
Respiratory Complexes ◽  
Stress Sensing ◽  
Mitochondrial Respiratory

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

scholarly journals An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

2020 ◽  
Vol 21 (3) ◽  
pp. 1074 ◽  
Author(s):  
Daniel Missailidis ◽  
Sarah J. Annesley ◽  
Claire Y. Allan ◽  
Oana Sanislav ◽  
Brett A. Lidbury ◽  
...  
Keyword(s):  
Steady State ◽  
Fatty Acid ◽  
Mitochondrial Function ◽  
Complex I ◽  
Atp Synthesis ◽  
Resting Cells ◽  
Respiratory Capacity ◽  
Respiratory Complexes ◽  
Stress Sensing ◽  
Mitochondrial Respiratory

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolizing patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signaling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters, and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

scholarly journals An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

2020 ◽  
Author(s):  
Daniel Missailidis ◽  
Sarah Annesley ◽  
Claire Allan ◽  
Oana Sanislav ◽  
Brett Lidbury ◽  
...  
Keyword(s):  
Steady State ◽  
Fatty Acid ◽  
Mitochondrial Function ◽  
Complex I ◽  
Atp Synthesis ◽  
Beta Oxidation ◽  
Respiratory Capacity ◽  
Respiratory Complexes ◽  
Stress Sensing ◽  
Mitochondrial Respiratory

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

scholarly journals An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

2019 ◽  
Author(s):  
Daniel Missailidis ◽  
Sarah Annesley ◽  
Claire Allan ◽  
Oana Sanislav ◽  
Brett Lidbury ◽  
...  
Keyword(s):  
Steady State ◽  
Fatty Acid ◽  
Mitochondrial Function ◽  
Complex I ◽  
Atp Synthesis ◽  
Beta Oxidation ◽  
Respiratory Capacity ◽  
Respiratory Complexes ◽  
Stress Sensing ◽  
Mitochondrial Respiratory

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by fatigue that is unaided by rest and by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”). There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by an elevation of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

scholarly journals Cryo-EM Studies of Respiratory Complexes in a Hyperthermophilic Archaeon Pyrococcus furiosus Suggest an Evolutionary Path to Modern-Day Complex I

2019 ◽  
Vol 25 (S2) ◽  
pp. 1354-1355
Author(s):  
Hongjun Yu ◽  
Gerrit J. Schut ◽  
Chang-Hao Wu ◽  
Dominik K. Haja ◽  
Michael W. Adams ◽  
...  
Keyword(s):  
Complex I ◽  
Pyrococcus Furiosus ◽  
Evolutionary Path ◽  
Hyperthermophilic Archaeon ◽  
Respiratory Complexes

scholarly journals Carbon and Nitrogen Sources Have No Impact on the Organization and Composition of Ustilago maydis Respiratory Supercomplexes

2021 ◽  
Vol 7 (1) ◽  
pp. 42
Author(s):  
Deyamira Matuz-Mares ◽  
Oscar Flores-Herrera ◽  
Guadalupe Guerra-Sánchez ◽  
Lucero Romero-Aguilar ◽  
Héctor Vázquez-Meza ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Atp Synthesis ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Energy Conditions ◽  
Respiratory Complexes ◽  
Respiratory Supercomplexes ◽  
Nadh Dehydrogenases ◽  
Reduced Nicotinamide Adenine Dinucleotide

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

scholarly journals Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria

1996 ◽  
Vol 318 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Susan K BUCHANAN ◽  
John E. WALKER
Keyword(s):  
Complex I ◽  
Large Scale ◽  
Gel Filtration ◽  
Atp Synthesis ◽  
Lipid Vesicles ◽  
Proton Pumping ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
F1fo Atpase

A new chromatographic procedure has been developed for the isolation of F1Fo-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl β-Δ-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1Fo-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351–357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50–80 mg of purified F1Fo-ATPase and 20–40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.

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