Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, L69 7ZB, UK; The Novo Nordisk Foundation Centre for Biosustainability, Technical University of Denmark, Building 220, Søltofts Plads, 2800 Kgs Lyngby, Denmark; Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch, Private Bag X1, Matieland, 7602, South Africa.
Biochim Biophys Acta Bioenerg. 2024 Nov 1;1865(4):149504. doi: 10.1016/j.bbabio.2024.149504. Epub 2024 Aug 15.
Two-stage (e.g. light-dark) phosphorylation experiments showed that there is a stored 'high-energy' intermediate linking electron transport and phosphorylation. Large, artificial electrochemical proton gradients (protonmotive forces or pmfs) can also drive phosphorylation, a fact seen as strongly supportive of the chemiosmotic coupling hypothesis that a pmf is the 'high-energy' intermediate. However, in such experiments there is an experimental threshold (pmf >170 mV, equivalent to ΔpH ∼2.8) below which no phosphorylation is in fact observed, and 220 mV are required to recreate in vivo rates. This leads to the correct question, which is then whether those values of the pmf generated by electron transport are large enough. Even the lower ones as required for any phosphorylation (leave alone those required to explain in vivo rates) are below the threshold [1, 2], whether measured directly with microelectrodes or via the use of membrane-permeant ions and/or acids/bases (which are always transporter substrates [3], so all such measurements are in fact artefactual). The single case that seemed large enough (220 mV) is now admitted to be a diffusion potential artefact [4]. Many other observables (inadequate bulk H in 'O-pulse'-type experiments, alkaliphilic bacteria, dual-inhibitor titrations, uncoupler-binding proteins, etc.) are consistent with the view that values of the pmf, and especially of Δψ, are actually very low. A protet-based charge separation model [2], a protonic version analogous to how energy may be stored in devices called electrets, provides a high-energy intermediate that can explain the entire literature, including the very striking demonstration [5] that close proximity is required between electron transport and ATP synthase complexes for energy coupling between them to allow phosphorylation to occur. A chief purpose of this article is thus to summarise the extensive and self-consistent literature, much of which is of some antiquity and rarely considered by modern researchers, despite its clear message of the inadequacy of chemiosmotic coupling to explain these phenomena.
两段式（例如，光照-黑暗）磷酸化实验表明，存在一个存储的“高能”中间产物，将电子传递和磷酸化联系起来。大的、人为的电化学质子梯度（质子动力或 pmf）也可以驱动磷酸化，这一事实强烈支持质子动力偶联假说，即 pmf 是“高能”中间产物。然而，在这样的实验中，存在一个实验阈值（pmf >170 mV，相当于 ΔpH ∼2.8），低于该阈值实际上观察不到磷酸化，而要重现体内速率则需要 220 mV。这就引出了一个正确的问题，即电子传递产生的 pmf 值是否足够大。即使是电子传递产生的任何磷酸化所需的较小值（更不用说解释体内速率所需的那些值）也低于阈值[1,2]，无论是直接用微电极测量还是通过使用膜通透离子和/或酸碱（它们始终是转运体的底物[3]，因此所有这些测量实际上都是人为的）。似乎足够大的单个案例（220 mV）现在被承认为扩散电位人为产物[4]。许多其他可观察到的现象（“O-脉冲”型实验中不足的细胞质 H、嗜碱细菌、双重抑制剂滴定、解偶联蛋白结合蛋白等）都与这样的观点一致，即 pmf 值，特别是 Δψ 值实际上非常低。一个基于质子的电荷分离模型[2]，类似于能量如何储存在称为驻极体的设备中的质子版本，提供了一个高能中间产物，可以解释整个文献，包括非常显著的演示[5]，即电子传递和 ATP 合酶复合物之间需要非常接近，才能使它们之间的能量偶联允许磷酸化发生。本文的主要目的之一是总结广泛而一致的文献，其中许多文献都具有一定的历史渊源，尽管它们清楚地表明质子动力偶联不足以解释这些现象，但现代研究人员很少考虑这些文献。
