Hitchens G D, Kell D B
Biochim Biophys Acta. 1984 Jul 27;766(1):222-32. doi: 10.1016/0005-2728(84)90235-4.
A fast-responding O2 electrode has been used to confirm and extend observations of a significant kinetic discrepancy between O2 reduction and consequent proton translocation in 'O2-pulse' experiments in intact cells of P. denitrificans. The permeant, chaotropic SCN- ion abolishes this discrepancy, and greatly increases the observable----H+/O ratio, to a value approaching its accepted, true, limiting stoichiometry. The observable H+ decay rates are very slow, particularly in the absence of SCN-. The submaximal----H+/O ratios observed in the absence of SCN- are essentially independent of the size of the O2 pulse, in a manner not easily explained by a delocalised chemiosmotic energy-coupling scheme. Osmotically active protoplasts of P. denitrificans do not show a significant kinetic discrepancy between O2 reduction and H+ translocation, even in the the absence of SCN-. However, the submaximal----H+/O ratios observed in the absence of SCN- are again essentially independent of the size of the O2 pulse. As in intact cells, the observable H+ decay rates are very slow. The energy-transfer inhibitor venturicidin causes a significant increase in the----H+/O ratio observed in protoplasts of P. denitrificans in the absence of SCN-; the decay kinetics of the H+ translocation process are also somewhat modified. Nevertheless, the----H+/O ratio observed in the presence of venturicidin is also independent of the size of the O2 pulse. This observation militates further against arguments in which (a) a non-ohmic leak of protons from the bulk aqueous phase might alone be the cause of the low----H+/O ratios observed in the absence of SCN-, and (b) in which there might be a delta p-dependent change ('redox slip') in the actual----H+/O ratio. It is concluded that the observable protonmotive activity of the respiratory chain of P. denitrificans in the absence of SCN- is directly influenced by the state of the H+-ATP synthetase in the cytoplasmic membrane of this organism. We are unable to explain the data in terms of a model in which the putative protonmotive force may be acting to affect the----H+/O ratio. The possibility is considered that the delocalised bulk-to-bulk phase membrane potential set up in response to protonmotive activity is energetically insignificant.
一种快速响应的氧电极已被用于证实和拓展在反硝化假单胞菌完整细胞的“氧脉冲”实验中，氧还原与随后的质子转运之间存在显著动力学差异的观察结果。渗透性的离液剂硫氰酸根离子消除了这种差异，并极大地提高了可观测到的H⁺/O比值，使其接近公认的真实极限化学计量比。可观测到的H⁺衰减速率非常缓慢，尤其是在没有硫氰酸根离子的情况下。在没有硫氰酸根离子时观察到的亚最大H⁺/O比值基本上与氧脉冲的大小无关，这种方式难以用离域化学渗透能量偶联机制来解释。反硝化假单胞菌的渗透活性原生质体即使在没有硫氰酸根离子的情况下，氧还原与H⁺转运之间也没有显著的动力学差异。然而，在没有硫氰酸根离子时观察到的亚最大H⁺/O比值同样基本上与氧脉冲的大小无关。与完整细胞一样，可观测到的H⁺衰减速率非常缓慢。能量转移抑制剂抗霉素A会使在没有硫氰酸根离子的情况下反硝化假单胞菌原生质体中观察到的H⁺/O比值显著增加；H⁺转运过程的衰减动力学也有所改变。尽管如此，在抗霉素A存在下观察到的H⁺/O比值也与氧脉冲的大小无关。这一观察结果进一步反对了以下观点：（a）质子从本体水相的非欧姆泄漏可能单独导致在没有硫氰酸根离子时观察到的低H⁺/O比值；（b）实际的H⁺/O比值可能存在依赖于质子动力势的变化（“氧化还原滑移”）。得出的结论是，在没有硫氰酸根离子的情况下，反硝化假单胞菌呼吸链可观测到的质子动力活性直接受该生物体细胞质膜中H⁺ - ATP合成酶状态的影响。我们无法用一个模型来解释这些数据，在该模型中假定的质子动力势可能作用于影响H⁺/O比值。考虑到这样一种可能性，即响应质子动力活性而建立的离域本体到本体相膜电位在能量上是微不足道的。
