FUNCTIONAL-PROPERTIES OF THE QUINOL OXIDASE FROM ACIDIANUS-AMBIVALENSAND THE POSSIBLE CATALYTIC ROLE OF ITS ELECTRON-DONOR - STUDIES ON THE MEMBRANE-INTEGRATED AND PURIFIED ENZYME

The aa(3) quinol oxidase has been purified from the thermoacidophilic archaea Acidianus ambivalens as a three-redox-centers enzyme. The func tional properties of this oxidase both as purified and in its most int egral form (i.e. in native membranes and in intact cells) were investi gated by stopped-flow spectrophotometry. The results suggest that the enzyme interacts in vivo with a redox-active molecule, which favours t he electron entry via heme a and provides the fourth electron demanded for catalysis. We observe that the purified enzyme has two hemes with apparent redox potentials 215+/-20 mV and 415+/-20 mV at pH 5.4, show ing redox-Bohr effect, and a heme a(3)-Cu-B center with an affinity fo r carbon monoxide (K-a = 5.7x10(4) M-1 at 35 degrees C) much lower tha n that reported for the mammalian enzyme (K-a = 4x10(6) M-1 at 20 degr ees C). The reduction by dithionite is fast and monophasic when the qu inol oxidase is in the native membranes, whereas it is slow and biphas ic in the purified enzyme (with heme a(3) being reduced faster than he me a). The oxygen reaction of the reduced purified enzyme is fast (few milliseconds), but yields an intermediate (likely ferryl) clearly dif ferent from the fully oxidized enzyme. In contrast, the same reaction performed in intact cells leads to the fully oxidized enzyme. We postu late that caldariella quinol, the physiological electron donor, is in vivo tightly bound to the enzyme, providing the fourth redox active ce nter lacking in the purified enzyme.