title: Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles.
abstract: Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles ("proteobicelles") were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL = 24 mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30-60 nm with the thickness of bicelles of about 4 nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160 nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited "normal" reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.
authors.affiliation: Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia. Electronic address: musatov@saske.sk.
authors.forename: Andrey
authors.initials: A
authors.lastname: Musatov
authors.affiliation: Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Siposova
authors.affiliation: Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia.
authors.forename: Martina
authors.initials: M
authors.lastname: Kubovcikova
authors.affiliation: Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia; Department of Biochemistry, Institute of Chemistry, P. J. Safarik University in Kosice, Moyzesova 11, 040 01 Kosice, Slovakia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Lysakova
authors.affiliation: Department of Biochemistry, Institute of Chemistry, P. J. Safarik University in Kosice, Moyzesova 11, 040 01 Kosice, Slovakia.
authors.forename: Rastislav
authors.initials: R
authors.lastname: Varhac
mesh_terms: D000818:Animals; D002417:Cattle; D003902:Detergents / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry
chemical_list: D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D003576:Electron Transport Complex IV; D004134:Dimyristoylphosphatidylcholine
keywords: Aggregation state; Bicelles; Cytochrome c oxidase; DHPC; DMPC; Detergents