Kannt A, Ostermann T, Müller H, Ruitenberg M
Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Frankfurt am Main, Germany.
FEBS Lett. 2001 Aug 17;503(2-3):142-6. doi: 10.1016/s0014-5793(01)02719-3.
Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn(2+) ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn(2+) concentration. Half-inhibition of proton pumping was observed at Zn(2+)=75 microM corresponding to about 5-6 Zn(2+) ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn(2+). Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn(2+) correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn(2+) ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase.
通过对黑色脂质膜进行停流分光光度法质子泵浦测量和时间分辨电位测量相结合的方法，我们研究了Zn(2+)离子对反硝化副球菌细胞色素c氧化酶质子转移特性的影响。当锌被包裹在含有脂质体的细胞色素c氧化酶内部时，发现H/e化学计量比随着Zn(2+)浓度的增加而逐渐降低。在Zn(2+)=75 microM时观察到质子泵浦的半抑制，这对应于每个氧化酶分子约5-6个Zn(2+)离子。此外，加入Zn(2+)后，蛋白脂质体的呼吸控制率显著增加。在黑色脂质膜上进行的时间分辨电位测量表明，在存在Zn(2+)的情况下，电生相减慢，这与归因于从细胞质侧摄取质子和质子泵浦的相相对应。我们得出结论，Zn(2+)离子结合在细菌细胞色素c氧化酶的两个质子转移途径附近或内部。
