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Time‐resolved generation of a membrane potential by ba 3 cytochrome c oxidase from Thermus thermophilus
Author(s) -
Siletskiy S.,
Soulimane T.,
Azarki.,
Vygodina T.V.,
Buse G.,
Kaulen A.,
Konstantinov A.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01019-4
Subject(s) - thermus thermophilus , chemistry , cytochrome c oxidase , electron transport complex iv , cyanide , oxidase test , hydrogen peroxide , photochemistry , stereochemistry , enzyme , inorganic chemistry , biochemistry , escherichia coli , gene
ba 3 ‐type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash‐induced generation of a membrane potential by the enzyme has been studied in a μs/ms time scale with Ru(II)‐tris‐bipyridyl complex (RuBpy) as a photoreductant. Flash‐induced single electron reduction of the aerobically oxidized ba 3 by RuBpy results in two phases of membrane potential generation by the enzyme with τ values of about 20 and 300 μs at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba 3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H 2 O 2 with a second order rate constant of ∼2×10 3 M −1 s −1 . The data indicate that single electron reduction of ba 3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba 3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba 3 oxidase as compared to the oxidases of the aa 3 ‐type.