scholarly journals Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Inorganics ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 38
Author(s):  
Janik Telleria Marloth ◽  
Constanze Pinske
Keyword(s):  
Complex I ◽  
H2 Production ◽  
Proton Pumping ◽  
Fermentative Hydrogen Production ◽  
Iron Sulfur ◽  
Formate Hydrogenlyase ◽  
Respiratory Complex I ◽  
Fermentative Hydrogen ◽  
Pumping Activity ◽  
Higher Sensitivity

Fermentative hydrogen production by enterobacteria derives from the activity of the formate hydrogenlyase (FHL) complex, which couples formate oxidation to H2 production. The molybdenum-containing formate dehydrogenase and type-4 [NiFe]-hydrogenase together with three iron-sulfur proteins form the soluble domain, which is attached to the membrane by two integral membrane subunits. The FHL complex is phylogenetically related to respiratory complex I, and it is suspected that it has a role in energy conservation similar to the proton-pumping activity of complex I. We monitored the H2-producing activity of FHL in the presence of different concentrations of the protonophore CCCP. We found an inhibition with an apparent EC50 of 31 µM CCCP in the presence of glucose, a higher tolerance towards CCCP when only the oxidizing hydrogenase Hyd-1 was present, but a higher sensitivity when only Hyd-2 was present. The presence of 200 mM monovalent cations reduced the FHL activity by more than 20%. The Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) combined with CCCP completely inhibited H2 production. These results indicate a coupling not only between Na+ transport activity and H2 production activity, but also between the FHL reaction, proton import and cation export.

scholarly journals The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jan Pyrih ◽  
Vojtěch Žárský ◽  
Justin D. Fellows ◽  
Christopher Grosche ◽  
Dorota Wloga ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Complex I ◽  
Phaeodactylum Tricornutum ◽  
Tetrahymena Thermophila ◽  
Thalassiosira Pseudonana ◽  
Cellular Distribution ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Secondary Plastids ◽  
Respiratory Complex I

Abstract Background Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea. To date, the cellular distribution of these proteins is considered to be highly conserved with only a few exceptions. Results We searched for the genes of all members of the Nbp35-like protein family and analyzed their targeting sequences. Nbp35 and Cfd1 were predicted to reside in the cytoplasm with some exceptions of Nbp35 localization to the mitochondria; Ind1was found in the mitochondria, and HCF101 was predicted to reside in plastids (chHCF101) of all photosynthetically active eukaryotes. Surprisingly, we found a second HCF101 paralog in all members of Cryptista, Haptista, and SAR that was predicted to predominantly target mitochondria (mHCF101), whereas Ind1 appeared to be absent in these organisms. We also identified a few exceptions, as apicomplexans possess mHCF101 predicted to localize in the cytosol and Nbp35 in the mitochondria. Our predictions were experimentally confirmed in selected representatives of Apicomplexa (Toxoplasma gondii), Stramenopila (Phaeodactylum tricornutum, Thalassiosira pseudonana), and Ciliophora (Tetrahymena thermophila) by tagging proteins with a transgenic reporter. Phylogenetic analysis suggested that chHCF101 and mHCF101 evolved from a common ancestral HCF101 independently of the Nbp35/Cfd1 and Ind1 proteins. Interestingly, phylogenetic analysis supports rather a lateral gene transfer of ancestral HCF101 from bacteria than its acquisition being associated with either α-proteobacterial or cyanobacterial endosymbionts. Conclusion Our searches for Nbp35-like proteins across eukaryotic lineages revealed that SAR, Haptista, and Cryptista possess mitochondrial HCF101. Because plastid localization of HCF101 was only known thus far, the discovery of its mitochondrial paralog explains confusion regarding the presence of HCF101 in organisms that possibly lost secondary plastids (e.g., ciliates, Cryptosporidium) or possess reduced nonphotosynthetic plastids (apicomplexans).

scholarly journals Functional Water Wires Catalyze Long-Range Proton Pumping in the Mammalian Respiratory Complex I

2020 ◽  
Vol 142 (52) ◽  
pp. 21758-21766
Author(s):  
Michael Röpke ◽  
Patricia Saura ◽  
Daniel Riepl ◽  
Maximilian C. Pöverlein ◽  
Ville R. I. Kaila
Keyword(s):  
Long Range ◽  
Complex I ◽  
Proton Pumping ◽  
Respiratory Complex ◽  
Respiratory Complex I

The coupling mechanism of mammalian respiratory complex I

Science ◽  
2020 ◽  
Vol 370 (6516) ◽  
pp. eabc4209 ◽  
Author(s):  
Domen Kampjut ◽  
Leonid A. Sazanov
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Pumping ◽  
State Transitions ◽  
Coupling Mechanism ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Respiratory Complex I

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo–electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

scholarly journals The multitude of iron–sulfur clusters in respiratory complex I

2016 ◽  
Vol 1857 (8) ◽  
pp. 1068-1072 ◽  
Author(s):  
Emmanuel Gnandt ◽  
Katerina Dörner ◽  
Marc F.J. Strampraad ◽  
Simon de Vries ◽  
Thorsten Friedrich
Keyword(s):  
Complex I ◽  
Iron Sulfur Clusters ◽  
Respiratory Complex ◽  
Iron Sulfur ◽  
Respiratory Complex I

Mammalian Respiratory Complex I Through the Lens of Cryo-EM

2019 ◽  
Vol 48 (1) ◽  
pp. 165-184 ◽  
Author(s):  
Ahmed-Noor A. Agip ◽  
James N. Blaza ◽  
Justin G. Fedor ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Respiratory Complex ◽  
Data Collection Strategy ◽  
Structural Work ◽  
Membrane Bound ◽  
Respiratory Complex I

Single-particle electron cryomicroscopy (cryo-EM) has led to a revolution in structural work on mammalian respiratory complex I. Complex I (mitochondrial NADH:ubiquinone oxidoreductase), a membrane-bound redox-driven proton pump, is one of the largest and most complicated enzymes in the mammalian cell. Rapid progress, following the first 5-Å resolution data on bovine complex I in 2014, has led to a model for mouse complex I at 3.3-Å resolution that contains 96% of the 8,518 residues and to the identification of different particle classes, some of which are assigned to biochemically defined states. Factors that helped improve resolution, including improvements to biochemistry, cryo-EM grid preparation, data collection strategy, and image processing, are discussed. Together with recent structural data from an ancient relative, membrane-bound hydrogenase, cryo-EM on mammalian complex I has provided new insights into the proton-pumping machinery and a foundation for understanding the enzyme's catalytic mechanism.

Measurement of H2 consumption and its role in continuous fermentative hydrogen production

2008 ◽  
Vol 57 (5) ◽  
pp. 681-685 ◽  
Author(s):  
J. T. Kraemer ◽  
D. M. Bagley
Keyword(s):  
Continuous Flow ◽  
H2 Production ◽  
Continuous Reactor ◽  
Yield Increase ◽  
Fermentative Hydrogen Production ◽  
Consumption Rates ◽  
Fermentative Hydrogen ◽  
H2 Yield ◽  
First Time

To maximise the yield from fermentative H2 production, H2 consumption must be minimised. This work demonstrated for the first time that H2 consumption exists in an established continuous-flow biohydrogen system. The rate of H2 consumption was found to be related to the concentration of CO2, with H2 consumption inhibited at both low and high CO2. N2 sparging of the continuous reactor at 31 mL/min/L-liquid increased the H2 yield from 1.31 to 1.87 mol H2/mol glucose, but did not significantly change the in-situ rate of H2 consumption (0.07–0.09 mM/h). Assuming sparging completely inhibited H2 consumption, it could only account for 2–11% of the H2 yield increase during sparging, based on H2 consumption rates measured in the reactor and in vials. Therefore, H2 consumption may be of minor concern for continuous biohydrogen systems.

scholarly journals Cryo-EM structure of respiratory complex I at work

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Kristian Parey ◽  
Ulrich Brandt ◽  
Hao Xie ◽  
Deryck J Mills ◽  
Karin Siegmund ◽  
...  
Keyword(s):  
Complex I ◽  
Atp Synthesis ◽  
Proton Pumping ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
Membrane Protein Complex ◽  
Change Mechanism ◽  
Steady State Activity ◽  
Respiratory Complex I ◽  
Ubiquinone Binding

Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.

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