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
A. Giuffre et al., 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, European journal of biochemistry, 250(2), 1997, pp. 383-388
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.