Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.
SpectroLogix LLC, 9050 Executive Park Drive, Knoxville, TN 37923, USA.
Biochim Biophys Acta Bioenerg. 2021 Mar 1;1862(3):148354. doi: 10.1016/j.bbabio.2020.148354. Epub 2020 Dec 16.
Cyclic electron flow (CEF) around photosystem I is vital to balancing the photosynthetic energy budget of cyanobacteria and other photosynthetic organisms. The coupling of CEF to proton pumping has long been hypothesized to occur, providing proton motive force (PMF) for the synthesis of ATP with no net cost to [NADPH]. This is thought to occur largely through the activity of NDH-1 complexes, of which cyanobacteria have four with different activities. While a much work has been done to understand the steady-state PMF in both the light and dark, and fluorescent probes have been developed to observe these fluxes in vivo, little has been done to understand the kinetics of these fluxes, particularly with regard to NDH-1 complexes. To monitor the kinetics of proton pumping in Synechocystis sp. PCC 6803, the pH sensitive dye Acridine Orange was used alongside a suite of inhibitors in order to observe light-dependent proton pumping. The assay was demonstrated to measure photosynthetically driven proton pumping and used to measure the rates of proton pumping unimpeded by dark ΔpH. Here, the cyanobacterial NDH-1 complexes are shown to pump a sizable portion of proton flux when CEF-driven and LEF-driven proton pumping rates are observed and compared in mutants lacking some or all NDH-1 complexes. It is also demonstrated that PSII and LEF are responsible for the bulk of light induced proton pumping, though CEF and NDH-1 are capable of generating ~40% of the proton pumping rate when LEF is inactivated.
循环电子流（CEF）绕过光系统 I 对于平衡蓝藻和其他光合生物的光合作用能量预算至关重要。CEF 与质子泵浦的偶联长期以来一直被假设为发生，为 ATP 的合成提供质子动力势（PMF），而不会对 [NADPH] 造成净成本。这被认为主要通过 NDH-1 复合物的活性来发生，其中蓝藻有四个具有不同活性的 NDH-1 复合物。虽然已经做了大量工作来了解在光照和黑暗中的稳态 PMF，并且已经开发了荧光探针来观察这些通量在体内的情况，但对于这些通量的动力学，特别是关于 NDH-1 复合物，几乎没有做过任何研究。为了监测 Synechocystis sp. PCC 6803 中的质子泵送动力学，使用了 pH 敏感染料吖啶橙，并结合了一系列抑制剂，以观察依赖光的质子泵送。该测定法被证明可测量光合作用驱动的质子泵送，并用于测量不受黑暗 ΔpH 阻碍的质子泵送速率。在这里，当观察和比较缺乏某些或所有 NDH-1 复合物的突变体中的 CEF 驱动和 LEF 驱动质子泵送速率时，显示出蓝藻 NDH-1 复合物在 CEF 驱动时泵送相当一部分质子通量。还证明 PSII 和 LEF 负责大部分光诱导的质子泵送，尽管当 LEF 失活时，CEF 和 NDH-1 能够产生 ~40%的质子泵送速率。
