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Biochim Biophys Acta. 2016 Dec;1858(12):2984-2992. doi: 10.1016/j.bbamem.2016.09.004. Epub 2016 Sep 13.

Isolation of yeast complex IV in native lipid nanodiscs.

Author information

1
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.
2
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
3
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
4
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.
5
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
6
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: peterb@dbb.su.se.

Abstract

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 bc1 in the mitochondrial membrane, cyt. bc1 was 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-1 and the kinetics of the reaction of the fully reduced CytcO with O2 was 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.

KEYWORDS:

Bioenergetics; Energy conservation; Membrane protein; Proton transfer

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