Siletskiy S, Soulimane T, Azarkina N, Vygodina T V, Buse G, Kaulen A, Konstantinov A
A.N. Belozerskiy Institute of Physico-Chemical Biology, Moscow State University, Russia.
FEBS Lett. 1999 Aug 20;457(1):98-102. doi: 10.1016/s0014-5793(99)01019-4.
ba3-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 microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 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 H2O2 with a second order rate constant of approximately 2 x 10(3) M-1 s-1. The data indicate that single electron reduction of ba3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba3 oxidase as compared to the oxidases of the aa3-type.
从嗜热栖热菌中纯化得到的 ba3 型细胞色素 c 氧化酶已被重组到磷脂囊泡中，并且在微秒/毫秒时间尺度上，以 Ru(II)-三联吡啶配合物（RuBpy）作为光还原剂，研究了该酶激光闪光诱导产生膜电位的情况。RuBpy 对需氧氧化的 ba3 进行闪光诱导的单电子还原，导致该酶产生两个膜电位生成阶段，在 pH 8 和 23 摄氏度时，时间常数 τ 值分别约为 20 和 300 微秒。分光光度实验表明，氧化态的 ba3 与过氧化氢或所研究的任何其他外源性血红素铁配体（如氰化物、硫化物和叠氮化物）反应很差。同时，RuBpy 对该酶的光还原引发了与 H2O2 的电化学反应，二级反应速率常数约为 2×10³ M⁻¹ s⁻¹。数据表明，ba3 氧化酶的单电子还原为外源性配体打开了该酶的双核中心。过氧化物在细胞色素 ba3 中诱导的质子电生相的分数贡献远小于在牛氧化酶中的贡献，这表明与 aa3 型氧化酶相比，ba3 氧化酶可能存在不同的电生机制。
