Tài liệu Báo cáo khoa học: Preliminary molecular characterization and crystallization of

Preliminary molecular characterization and crystallization

of mitochondrial respiratory complex II from porcine heart

Xia Huo1,2, Dan Su1

, Aojin Wang2

, Yujia Zhai2

, Jianxing Xu2

, Xuemei Li2

, Mark Bartlam1,2,

Fei Sun2 and Zihe Rao1,2

1 Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing, China

2 National Laboratory of Biomacromolecules, Institute of Biophysics (IBP), Chinese Academy of Sciences, Beijing, China

Mitochondria are cellular organelles of prokaryotic

origin that are found in almost all eukaryotic cells.

The mitochondrial respiratory system, consisting of

five membrane protein complexes (I–V), produces most

of the energy in eukaryotic cells by a process called

oxidative phosphorylation [1]. Electrons are passed

along a series of respiratory enzyme complexes located

in the inner mitochondrial membrane, and the energy

released by this electron transfer is used to pump pro￾tons across the inner membrane.

The electron transport chain comprises five respira￾tory enzyme complexes arranged in a specific orienta￾tion. Complex I (NADH:ubiquinone oxidoreductase),

the largest and most complicated of these, is the main

point of entry into the respiratory chain for electrons.

The mammalian complex I contains nearly 50 different

subunits in an unknown stoichiometry [2–5]. Com￾plex II (succinate:ubiquinone oxidoreductase) is a com￾ponent of the tricarboxylic acid cycle (Krebs cycle),

and participates in the electron transport chain by

transferring electrons from succinate to the ubiquinone

pool. Complex III (cytochrome bc1 complex) delivers

electrons from ubiquinone to cytochrome c. It cou￾ples this redox reaction to the generation of a proton

gradient across the membrane by a mechanism known

as the Q cycle [1,6]. Complex IV (cytochrome c oxid￾ase complex), as the terminal enzyme of biological oxi￾dation, catalyzes the reduction of O2 to H2O at the

Keywords

complex II; crystallization; membrane

protein; mitochondrial respiration;

sequencing

Correspondence

M. Bartlam, Laboratory of Structural

Biology, Life Sciences Building, Tsinghua

University, Beijing 100084, China

Fax: +86 10 62773145

Tel: +86 10 62771493

E-mail: [email protected]

F. Sun, Laboratory of Biological Electron

Microscopy and Structural Biology, Institute

of Biophysics, Chinese Academy of

Sciences, 15 Datun Road, Chaoyang

District, Beijing 100101, China

Fax ⁄ Tel: +86 10 64888582

E-mail: [email protected]

(Received 10 November 2006, revised 18

December 2006, accepted 15 January 2007)

doi:10.1111/j.1742-4658.2007.05698.x

The mitochondrial respiratory complex II, or succinate:ubiquinone oxido￾reductase, is an integral membrane protein complex in both the tricarboxy￾lic acid cycle (Krebs cycle) and aerobic respiration. The gene sequences of

each complex II subunit were measured by RT-PCR. N-terminal sequen￾cing work was performed to identify the mitochondrial targeting signal

peptide of each subunit. Complex II was extracted from porcine heart and

purified by the ammonium sulfate precipitation method. The sample was

solubilized by 0.5% (w ⁄ v) sugar detergent n-decyl-b-d-maltoside, stabilized

by 200 mm sucrose, and crystallized with 5% (w ⁄ v) poly(ethylene gly￾col) 4000. Important factors for the extraction, purification and crystalliza￾tion of mitochondrial respiratory complex II are discussed.

Abbreviations

Ip, iron-sulfur protein; Fp, flavoprotein.

1524 FEBS Journal 274 (2007) 1524–1529 ª 2007 The Authors Journal compilation ª 2007 FEBS