Na(+)-translocating cytochrome bo terminal oxidase from Vitreoscilla: some parameters of its Na+ pumping and orientation in synthetic vesicles.
Vitreoscilla cytochrome bo ubiquinol oxidase is similar in some properties to the Escherichia coli enzyme, but unlike the latter, the Vitreoscilla oxidase functions as a primary Na+ pump. When purified Vitreoscilla cytochrome bo is incorporated into liposomes made from Vitreoscilla phospholipids and energized with a quinol substrate, it translocates Na+, not H+, across the vesicle membrane. Since protonophores CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DTHB (3,5-di-tert-butyl-4-hydroxybenzaldehyde) stimulated the Na+ pumping, it is unlikely that it is a secondary effect due to the presence of Na+/H+ antiporter activity in the preparations. The efficiency of the Na+ pumping was 3.93 Na+ pumped per O2 consumed when ascorbate/TMPD was used as the substrate. The cytochrome has a K(m) and Kcat for Na+ of 2.9 mM and 277 s-1, respectively. When ferricytochrome c was entrapped within liposomes prepared from Vitreoscilla phospholipids, it was reduced by Q1H2 (ubiquinol-1) but not by ascorbate/TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine). Although Q1H2 was oxidized by cytochrome bo in solution at a rate approximately 14 times that of the latter substrate, the rate of accumulation of Na+ within cytochrome bo vesicles driven by the membrane impermeable ascorbate/TMPD was 1.23 times that of the membrane permeable ubiquinol. These data allowed a calculation that in these synthetic proteoliposomes the cytochrome bo molecules are only 51% directed inward; a value of 61% inward-directed was estimated by measuring the ascorbate/TMPD oxidase activity of the proteoliposomes before and after disrupting them with Triton X-100. A random orientation of the E. coli cytochrome bo oxidase in proteoliposomes has also been reported.
Park
C
C
Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago 60616, USA.
Moon
J Y
JY
Cokic
P
P
Webster
D A
DA
D001205:Ascorbic Acid / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010226:Parabens / Q000494:pharmacology; D010539:Permeability; D010655:Phenylenediamines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D007476:Ionophores; D008081:Liposomes; D010226:Parabens; D010655:Phenylenediamines; D010743:Phospholipids; D014451:Ubiquinone; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C017168:cytochrome bo, E coli; C062323:3,5-di-tert-butyl-4-hydroxybenzoic acid; D012964:Sodium; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D000254:Sodium-Potassium-Exchanging ATPase; C003741:ubiquinol; D001205:Ascorbic Acid; D010100:Oxygen