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

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

1988 ◽  
Vol 155 (3) ◽  
pp. 1145-1153 ◽  
Author(s):  
Romana Fato ◽  
Cinzia Castelluccio ◽  
Stefania Armaroli ◽  
Alberto Contarini ◽  
Giovanna Parenti Castelli ◽  
...  
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Submitochondrial Particles ◽  
Cytochrome C Reductase ◽  
Bovine Heart ◽  
Steady State Kinetics ◽  
Kinetics Of

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 The steady state kinetics of bovine heart NADH:coenzyme Q oxidoreductase

2003 ◽  
Vol 43 (supplement) ◽  
pp. S182
Author(s):  
N. Hano ◽  
Y. Nakashima ◽  
K. Shinzawa-Itoh ◽  
H. Terada ◽  
Yoshikawa Yoshikawa
Keyword(s):  
Steady State ◽  
Bovine Heart ◽  
Steady State Kinetics ◽  
Kinetics Of

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.

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