scholarly journals The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa

2003 ◽  
Vol 369 (3) ◽  
pp. 619-626 ◽  
Author(s):  
Vera G. GRIVENNIKOVA ◽  
Darya V. SEREBRYANAYA ◽  
Elena P. ISAKOVA ◽  
Tatyana A. BELOZERSKAYA ◽  
Andrei D. VINOGRADOV
Keyword(s):  
Steady State ◽  
Neurospora Crassa ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Nadh Oxidase ◽  
Paracoccus Denitrificans ◽  
Molecular Genetic ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Genetic Manipulations ◽  
Slow Cycling

The mammalian mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been shown to exist in two kinetically and structurally distinct slowly interconvertible forms, active (A) and de-activated (D) [Vinogradov and Grivennikova (2001) IUBMB Life 52, 129—134]. This work was undertaken to investigate the putative Complex I A—D transition in the mitochondrial membrane of the lower eukaryote Neurospora crassa and in plasma membrane of the prokaryote Paracoccus denitrificans, organisms that are eligible for molecular genetic manipulations. The potential interconversion between A and D forms was assessed by examination of the initial and steady-state rates of NADH oxidation catalysed by inside-out submitochondrial (N. crassa) and sub-bacterial (P. denitrificans) particles and their sensitivities to N-ethylmaleimide and Mg2+. All diagnostic tests provide evidence that slow temperature- and turnover-dependent A—D transition is an explicit feature of eukaryotic N. crassa Complex I, whereas the phenomenon is not seen in the membranes of the prokaryote P. denitrificans. Significantly lower activation energy for A-to-D transition characterizes the N. crassa enzyme compared with that determined previously for the mammalian Complex I. Either a lag or a burst in the onset of the NADH oxidase assayed in the presence of Mg2+ is seen when the reaction is initiated by the thermally de-activated or NADH-activated particles, whereas the delayed final activities of both preparations are the same. We conclude that continuous slow cycling between A and D forms occurs during the steady-state operation of Complex I in N. crassa mitochondria.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Owen D. Jarman ◽  
Olivier Biner ◽  
John J. Wright ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Paracoccus Denitrificans ◽  
Model System ◽  
Proton Pumping ◽  
Model Systems ◽  
Metabolic Enzyme ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
Inner Mitochondrial Membrane

AbstractMitochondrial 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.

scholarly journals Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

2015 ◽  
Vol 290 (34) ◽  
pp. 20761-20773 ◽  
Author(s):  
Shaotong Zhu ◽  
Steven B. Vik
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Nadh Oxidase ◽  
Membrane Surface ◽  
Proton Translocation ◽  
Membrane Vesicles ◽  
Peripheral Region ◽  
Significant Loss ◽  
Cysteine Residues ◽  
Nadh:Ubiquinone Oxidoreductase

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

scholarly journals Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

2007 ◽  
Vol 6 (12) ◽  
pp. 2391-2405 ◽  
Author(s):  
Isabel Marques ◽  
Norbert A. Dencher ◽  
Arnaldo Videira ◽  
Frank Krause
Keyword(s):  
Neurospora Crassa ◽  
Respiratory Chain ◽  
Complex I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Deletion Mutants ◽  
Supramolecular Organization ◽  
Wild Type ◽  
Respiratory Supercomplexes ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.

scholarly journals The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria

2003 ◽  
Vol 371 (3) ◽  
pp. 1005-1011 ◽  
Author(s):  
Margarida DUARTE ◽  
Markus PETERS ◽  
Ulrich SCHULTE ◽  
Arnaldo VIDEIRA
Keyword(s):  
Neurospora Crassa ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Point Mutations ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Prosthetic Group ◽  
Reading Frame ◽  
Mature Enzyme ◽  
Nadh Dehydrogenases

An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination.

scholarly journals Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

2015 ◽  
Vol 290 (34) ◽  
pp. 20815-20826 ◽  
Author(s):  
Mahmoud M. Elguindy ◽  
Eiko Nakamaru-Ogiso
Keyword(s):  
Complex I ◽  
Nadh Oxidase ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Knock Out ◽  
Enhancing Effect ◽  
Highly Sensitive ◽  
Death Effector ◽  
Reconstituted Membranes ◽  
Apoptosis Inducing Factor

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.

scholarly journals Primary structure and mitochondrial import in vitro of the 20.9 kDa subunit of complex I from Neurospora crassa

1992 ◽  
Vol 288 (1) ◽  
pp. 29-34 ◽  
Author(s):  
J E Azevedo ◽  
U Nehls ◽  
C Eckerskorn ◽  
H Heinrich ◽  
H Rothe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Primary Structure ◽  
Complex I ◽  
Striking Similarity ◽  
Mitochondrial Outer Membrane ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Import ◽  
Inner Membrane

The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic ribosomes lacking a cleavable targeting sequence, interacts with the mitochondrial outer membrane (in a process that does not require an energized inner membrane), and is imported into mitochondria at contact sites. The 20.9 kDa subunit is then inserted into the inner membrane acquiring a topology similar to that of the already assembled subunit.

scholarly journals Inhibitory effect of palmitate on the mitochondrial NADH:ubiquinone oxidoreductase (complex I) as related to the active–de-active enzyme transition

2005 ◽  
Vol 387 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Maria V. LOSKOVICH ◽  
Vera G. GRIVENNIKOVA ◽  
Gary CECCHINI ◽  
Andrei D. VINOGRADOV
Keyword(s):  
Oxidase Activity ◽  
Complex I ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
Residual Activity ◽  
Quinone Reductase ◽  
Enzyme Concentration ◽  
Complete Relief ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Activated Complex

Palmitate rapidly and reversibly inhibits the uncoupled NADH oxidase activity catalysed by activated complex I in inside-out bovine heart submitochondrial particles (IC50 extrapolated to zero enzyme concentration is equal to 9 μM at 25 °C, pH 8.0). The NADH:hexa-ammineruthenium reductase activity of complex I is insensitive to palmitate. Partial (∼50%) inhibition of the NADH:external quinone reductase activity is seen at saturating palmitate concentration and the residual activity is fully sensitive to piericidin. The uncoupled succinate oxidase activity is considerably less sensitive to palmitate. Only a slight stimulation of tightly coupled respiration with NADH as the substrate is seen at optimal palmitate concentrations, whereas complete relief of the respiratory control is observed with succinate as the substrate. Palmitate prevents the turnover-induced activation of the de-activated complex I (IC50 extrapolated to zero enzyme concentration is equal to 3 μM at 25 °C, pH 8.0). The mode of action of palmitate on the NADH oxidase is qualitatively temperature-dependent. Rapid and reversible inhibition of the complex I catalytic activity and its de-active to active state transition are seen at 25 °C, whereas the time-dependent irreversible inactivation of the NADH oxidase proceeds at 37 °C. Palmitate drastically increases the rate of spontaneous de-activation of complex I in the absence of NADH. Taken together, these results suggest that free fatty acids act as specific complex I-directed inhibitors; at a physiologically relevant temperature (37 °C), their inhibitory effects on mitochondrial NADH oxidation is due to perturbation of the pseudo-reversible active–de-active complex I transition.

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