Functional Properties of the Quinol Oxidase from Acidianus ambivalens and the Possible Catalytic Role of its Electron Donor

Abstract

The aa 3 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 215Ϯ 20 mV and 415 Ϯ20 mV at pH 5.4, showing redox-Bohr effect, and a heme a 3 -Cu B center with an affinity for carbon monoxide (K a ϭ 5.7ϫ104 M Ϫ1 at 35°C) much lower than that reported for the mammalian enzyme (K a ϭ 4ϫ10 6 M Ϫ1 at 20°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 a 3 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.

Acidianus ambivalens Complex II Typifies a Novel Family of Succinate Dehydrogenases

“…It is composed by a type-II NADH dehydrogenase, solely containing a covalently bound flavin as cofactor (26); a complex II with a different subunit and cofactor composition (this work); a pool of caldariella quinone (18), which carries electrons between the dehydrogenases and a single type of complex IV, an aa 3 -type quinol oxidase (27)(28)(29)(30).…”

The Confluence of Heavy Metal Biooxidation and Heavy Metal Resistance: Implications for Bioleaching by Extreme Thermoacidophiles

“…The SoxABCD-SoxL complex, an aa3 quinol oxidase [102][103][104], the SoxM supercomplex, a bb3 terminal oxidase [105][106][107][108], and the CbsAB-SoxLN complex, a cytochrome ba [109] of S. acidocaldarius, later found in A. ambivalens [110], and the DoxBCE complex, an aa3-type quinol oxidase [111][112][113] of A. ambivalens had encapsulated the current view of aerobic respiratory electron transfer in the Sulfolobales. Unfortunately, neither S. acidocaldarius nor A. ambivalens can grow well on metal sulfides, thus motivating study of Crenarchaeota capable of growth on metal sulfides.…”

Redox-linked transient deprotonation at the binuclear site in the aa 3 -type quinol oxidase from Acidianus ambivalens : Implications for proton translocation

“…Recently, the kinetics of the electron transfer steps in this enzyme were studied, and a physiological role for the membrane-bound quinol serving as the fourth redox center was suggested (5). In the present study, the aa 3 quinol oxidase from A. ambivalens has been characterized by resonance Raman scattering, a technique that has been shown to be very powerful in studying heme-copper oxidases (13,(21)(22)(23).…”

“…On the contrary, the membrane-integrated enzyme has a bound quinol (5). When the reduction of the membrane fraction or the detergent-solubilized membranes was carried out under similar conditions as used for the purified enzyme, the 1,667-cm Ϫ1 line appeared within our 1-min limit of reducing the enzyme and measuring a spectrum (spectra not shown, data listed in Table 1).…”

“…and M.T., unpublished data). The a-type hemes belong to a single aa 3 -type quinol oxidase (1,5), which is the only heme protein present in the membranes, and contain a hydroxyethylgeranylgeranyl side chain instead of a hydroxyethylfarnesyl side chain as seen in most bacterial and eukaryotic aa 3 oxidases (6). The major quinone in aerobically grown A. ambivalens is caldariella quinone, which is a thiophenobenzoquinone present in the Sulfolobales (7).…”