Characterization of a mutation that abolishes quinone reduction by electron transfer flavoprotein-ubiquinone oxidoreductase

1995 ◽  
Vol 4 (2) ◽  
pp. 157-161 ◽  
Author(s):  
Shannon E. Beard ◽  
Stephen I. Goodman ◽  
Kristina Bemelen ◽  
Frank E. Frerman
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Quinone Reduction

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein

2002 ◽  
Vol 364 (3) ◽  
pp. 659-667 ◽  
Author(s):  
Martin ŠIMKOVIČ ◽  
Gregory D. DEGALA ◽  
Sandra S. EATON ◽  
Frank E. FRERMAN
Keyword(s):  
Electron Transfer ◽  
Expression System ◽  
Active Form ◽  
Integral Membrane Protein ◽  
Redox Potentials ◽  
Baculovirus Vector ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Steady State Kinetic ◽  
Catalytically Active

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron—sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron—sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus—human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron—sulphur flavoprotein.

scholarly journals Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein

1997 ◽  
Vol 272 (42) ◽  
pp. 26425-26433 ◽  
Author(s):  
Denise Salazar ◽  
Lening Zhang ◽  
Gregory D. deGala ◽  
Frank E. Frerman
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Flavoprotein ◽  
Pathogenic Mutations

Systemic carnitine deficiency due to lack of electron transfer flavoprotein: ubiquinone oxidoreductase

Neurology ◽  
1986 ◽  
Vol 36 (7) ◽  
pp. 957-957 ◽  
Author(s):  
S. D. Donato ◽  
F. E. Frerman ◽  
M. Rimoldi ◽  
P. Rinaldo ◽  
F. Taroni ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Carnitine Deficiency ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Systemic Carnitine Deficiency

scholarly journals The Iron−Sulfur Cluster of Electron Transfer Flavoprotein−Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein†

Biochemistry ◽  
2008 ◽  
Vol 47 (34) ◽  
pp. 8894-8901 ◽  
Author(s):  
Michael A. Swanson ◽  
Robert J. Usselman ◽  
Frank E. Frerman ◽  
Gareth R. Eaton ◽  
Sandra S. Eaton
Keyword(s):  
Electron Transfer ◽  
Electron Acceptor ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Iron Sulfur
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