Isolation of yeast complex IV in native lipid nanodiscs.
We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11nm×14nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc1in the mitochondrial membrane, cyt. bc1was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O2-reduction activity of ~130 electrons CytcO-1s-1and the kinetics of the reaction of the fully reduced CytcO with O2was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden; Belozersky Institute, Moscow State University, Leninskie Gory 1, Bldg. 40, 119991 Moscow, Russian Federation.
Irina A
IA
Smirnova
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Dan
D
Sjöstrand
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Fei
F
Li
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Markus
M
Björck
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Jacob
J
Schäfer
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Henrik
H
Östbye
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden; Department of Chemistry, Stanford University, Stanford, California 94305, United States.
Martin
M
Högbom
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
Christoph
C
von Ballmoos
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Gabriel C
GC
Lander
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Pia
P
Ädelroth
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: peterb@dbb.su.se.
Peter
P
Brzezinski
D020134:Catalytic Domain; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification*; D008055:Lipids / Q000032:analysis*; D008298:Maleates / Q000494:pharmacology; D053758:Nanoparticles; D011137:Polystyrenes / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D008055:Lipids; D008298:Maleates; D011137:Polystyrenes; C024074:styrene-maleic acid polymer; D003576:Electron Transport Complex IV
Bioenergetics; Energy conservation; Membrane protein; Proton transfer