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

ABSTRACT: The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme or the purified enzyme preincubated with decylubiquinol, this mode appears at 1,667 cm(-1) much more rapidly, suggesting a role of the bound quinol in controlling the redox-linked conformational changes. The shift of the formyl mode to higher frequency is attributed to a loss of hydrogen bonding that is associated with a group having a pKa of approximately 3.8. Based on these observations, a crucial element for proton translocation involving a redox-linked conformational change near the heme a(3) formyl group is postulated.

SUBMITTER: Das TK

PROVIDER: S-EPMC22253 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature

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

Das T K TK Gomes C M CM Teixeira M M Rousseau D L DL

Proceedings of the National Academy of Sciences of the United States of America 19990801 17

The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme ...[more]

PMID: 10449737

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Project description:BackgroundThe thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO(2)-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea.ResultsA. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 10(8)/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent.ConclusionThe soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Dataset Information

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

Publications

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

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