The Flavoprotein Subcomplex of Complex I (NADH:Ubiquinone Oxidoreductase) from Bovine Heart Mitochondria:  Insights into the Mechanisms of NADH Oxidation and NAD+Reduction from Protein Film Voltammetry†

Biochemistry ◽  
2007 ◽  
Vol 46 (11) ◽  
pp. 3454-3464 ◽  
Author(s):  
Chérise D. Barker ◽  
Torsten Reda ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Nadh Oxidation ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Bovine Heart

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.

scholarly journals Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

2006 ◽  
Vol 400 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Steven Sherwood ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Active Sites ◽  
Proton Translocation ◽  
Energy Transduction ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Protonmotive Force ◽  
Bovine Heart ◽  
Quinone Reduction ◽  
Q Cycle

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the membrane-bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a protonmotive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron–sulfur clusters transferring electrons between the two active sites. Here, we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc1 complex. No evidence for the ‘reductant-induced oxidation’ of ubiquinol could be detected; therefore no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.

scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits

1991 ◽  
Vol 278 (3) ◽  
pp. 821-829 ◽  
Author(s):  
I M Fearnley ◽  
M Finel ◽  
J M Skehel ◽  
J E Walker
Keyword(s):  
Amino Acid ◽  
Protein Sequence ◽  
Complex I ◽  
Mitochondrial Gene ◽  
Amino Acid Sequences ◽  
Heart Mitochondria ◽  
Blue Dye ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Cdna Clones ◽  
Bovine Heart

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit.

Steady-State Kinetics of the Reduction of Coenzyme Q Analogs by Complex I (NADH:Ubiquinone Oxidoreductase) in Bovine Heart Mitochondria and Submitochondrial Particles†

Biochemistry ◽  
1996 ◽  
Vol 35 (8) ◽  
pp. 2705-2716 ◽  
Author(s):  
Romana Fato ◽  
Ernesto Estornell ◽  
Salvatore Di Bernardo ◽  
Francesco Pallotti ◽  
Giovanna Parenti Castelli ◽  
...  
Keyword(s):  
Steady State ◽  
Complex I ◽  
Coenzyme Q ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Steady State Kinetics ◽  
Kinetics Of

scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment

FEBS Letters ◽  
1991 ◽  
Vol 282 (1) ◽  
pp. 135-138 ◽  
Author(s):  
J.Mark Skehel ◽  
Stephanie J. Pilkington ◽  
Michael J. Runswick ◽  
Ian M. Fearnley ◽  
John E. Walker
Keyword(s):  
Dna Sequence ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Complementary Dna ◽  
Bovine Heart

scholarly journals Characterization of an NADH-dependent haem-degrading system in ox heart mitochondria

1987 ◽  
Vol 246 (2) ◽  
pp. 467-474 ◽  
Author(s):  
R K Kutty ◽  
M D Maines
Keyword(s):  
Cytochrome C ◽  
Complex I ◽  
Effective Interaction ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Reaction Products ◽  
Degradation Activity ◽  
Haem Oxygenase ◽  
Biliverdin Ix ◽  
Degrading System

We report the identification of an NADH-dependent haem-degrading system in ox heart mitochondria. The activity was localized to the mitochondrial inner membrane, specifically associated with complex I (NADH:ubiquinone oxidoreductase). The mitochondrial NADH-dependent haem-degradation activity was highly effective and displayed a rate nearly 60% higher than that of the microsomal activity. The following observations suggested the enzymic nature of the activity: (i) haem degradation by complex I did not proceed upon exposure to elevated temperature and extremes of pH; (ii) it displayed substrate specificity; (iii) it was inhibited by a substrate analogue; and (iv) it showed a cofactor requirement. Moreover, the activity was distinctly different from the ascorbate-mediated haem-degradation activity. Also, complex I differed from the microsomal NADPH:cytochrome c (P-450) reductase inasmuch as the formation of an effective interaction with the microsomal haem oxygenase could not be detected. Addition of purified haem oxygenase to complex I neither influenced the rate of haem degradation nor resulted in the formation of biliverdin IX alpha. In contrast, addition of haem oxygenase to NADPH:cytochrome c (P-450) reductase enhanced the rate of haem degradation by nearly 8-fold, and more than 60% of the degraded haem could be accounted for as biliverdin IX alpha. The haem-degrading activity of complex I appeared to involve the activity of H2O2, as the reaction was inhibited by nearly 90% by catalase, and propentdyopents were detected as reaction products. Intact haemoproteins such as cytochrome c and myoglobin were not effective substrates. However, the haem undecapeptide of cytochrome c was degraded at a rate equal to that observed for haem. Haematohaem was degraded at a rate 50% lower than that observed for haem. It is suggested that the NADH-dependent haem-degradation system may have a biological role in the regulation of the concentration of respiratory haemoproteins and the disposition of the aberrant forms of the mitochondrial haemoproteins.

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