Biophysical and biochemical characterization of reconstituted and purified Rhodobacter sphaeroides cytochrome c oxidase in phospholipid vesicles sheds insight into its functional oligomeric structure.
Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.
Cvetkov
Teresa L
TL
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine and College of Science and Mathematics, Wright State University, Dayton, OH 45435, USA.
Prochaska
Lawrence J
LJ
D055592:Biophysical Phenomena; D001703:Biophysics; D002499:Centrifugation, Density Gradient; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D008081:Liposomes; D010743:Phospholipids; D011522:Protons; D003576:Electron Transport Complex IV