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 proper ties to the Escherichia coli enzyme, but unlike the latter, the Vitreo scilla oxidase functions as a primary Na+ pump. When purified Vitreosc illa cytochrome bo is incorporated into liposomes made from Vitreoscil la phospholipids and energized with a quinol substrate, it translocate s 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 act ivity in the preparations. The efficiency of the Na+ pumping was 3.93 Na+ pumped per O-2 consumed when ascorbate/TMPD was used as the substr ate. The cytochrome has a K-m and k(cat) for Na+ of 2.9 mM and 277 s(- 1), respectively. When ferricytochrome c was entrapped within liposome s prepared from Vitreoscilla phospholipids, it was reduced by Q(1)H(2) (ubiquinol-1) but not by ascorbate/TMPD (N,N,N',N'-tetramethyl-1,4-ph enylenediamine). Although Q(1)H(2) was oxidized by cytochrome bo in so lution at a rate approximately 14 times that of the latter substrate, the rate of accumulation of Na+ within cytochrome bo vesicles driven b y the membrane impermeable ascorbate/TMPD was 1.23 times that of the m embrane permeable ubiquinol. These data allowed a calculation that in these synthetic proteoliposomes the cytochrome bo molecules are only 5 1% directed inward; a value of 61% inward-directed was estimated by me asuring the ascorbate/TMPD oxidase activity of the proteoliposomes bef ore and after disrupting them with Triton X-100. A random orientation of the E. coli cytochrome bo oxidase in proteoliposomes has also been reported.