Functional properties of the quinol oxidase from Acidianus ambivalens and the possible catalytic role of its electron donor--studies on the membrane-integrated and purified enzyme.
The aa3 quinol oxidase has been purified from the thermoacidophilic archaea Acidianus ambivalens as a three-redox-centers enzyme. The functional properties of this oxidase both as purified and in its most integral form (i.e. in native membranes and in intact cells) were investigated by stopped-flow spectrophotometry. The results suggest that the enzyme interacts in vivo with a redox-active molecule, which favours the 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, showing redox-Bohr effect, and a heme a3-CuB center with an affinity for carbon monoxide (Ka = 5.7 x 10(4) M(-1) at 35 degrees C) much lower than that reported for the mammalian enzyme (Ka = 4 x 10(6) M(-1) at 20 degrees C). The reduction by dithionite is fast and monophasic when the quinol oxidase is in the native membranes, whereas it is slow and biphasic in the purified enzyme (with heme a3 being reduced faster than heme a). The oxygen reaction of the reduced purified enzyme is fast (few milliseconds), but yields an intermediate (likely ferryl) clearly different from the fully oxidized enzyme. In contrast, the same reaction performed in intact cells leads to the fully oxidized enzyme. We postulate that caldariella quinol, the physiological electron donor, is in vivo tightly bound to the enzyme, providing the fourth redox active center lacking in the purified enzyme.
Giuffrè
A
A
Department of Biochemical Sciences and CNR Center of Molecular Biology, University of Rome La Sapienza, Italy.
Gomes
C M
CM
Antonini
G
G
D'Itri
E
E
Teixeira
M
M
Brunori
M
M
D004227:Dithionite / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D016943:Sulfolobaceae / Q000201:enzymology*
D004227:Dithionite; D010088:Oxidoreductases; C020094:duroquinol oxidase; D010100:Oxygen