http://www.real-times.com.cn/guest/20143219541398.pdf

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

http://science.sciencemag.org/content/sci/272/5265/1136.full.pdf

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.

https://science.sciencemag.org/content/sci/269/5227/1069.full.pdf

Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A

Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea

Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2881%2980932-5

On the function of multiple subunits of cytochrome c oxidase from higher eukaryotes.

Cytochrome c oxidase from rat liver mitochondria was separated into 12 different protein subunits by application of a highly resolving sodium dodecylsulfate/gel electrophoretic system of different compositions. The 12 protein subunits are shown to represent integral components of mammalian type cytochrome c oxidase for the following reasons. 1. All 12 subunits copurify through various purification procedures. 2. The subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. 3. All 12 subunits are present in the complex at one to one stoichiometric amounts. 4. A similar composition of 12 subunits was also found for cytochrome c oxidase from rat kidney, pig heart, rabbit liver and stone-marten liver. The difference between our results and all other published data on the subunit composition of mammalian-type cytochrome c oxidase, based on gel electrophoretic analysis, is due to the insufficient resolving power of previously used gel systems and the very similar molecular weight of subunits VIa, b, c, and VIIa, b, c.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb04525.x

The Subunit Composition of Mammalian Cytochrome c Oxidase

1. The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eightfold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content. 2. The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c-depleted membranes, deoxycholate-dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low-affinity and for the high-affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes. 3. Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme. 4. By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase isoenzymes in liver and heart is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1982.tb06674.x

Peter Merle

Papers

Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure.

On the function of multiple subunits of cytochrome c oxidase from higher eukaryotes.

The Subunit Composition of Mammalian Cytochrome c Oxidase

Kinetic and structural differences between cytochrome c oxidases from beef liver and heart.

Immunological and chemical characterization of rat liver cytochrome c oxidase