Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer
Charge Regulation in a Rieske Proton Pump Pinpoints Zero, One, and Two Proton-Coupled Electron Transfer
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DOI：
10.1021/jacs.3c03006
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发表时间：
2023-07-24
影响因子：
15
通讯作者：
Shaw，Bryan F. F.
中科院分区：
文献类型：
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作者：
Koone，Jordan C. C.;Simmang，Mikaela;Shaw，Bryan F. F.
由于测量溶剂化蛋白质的净电荷存在困难，氧化还原驱动的质子泵在电子转移过程中调节净电荷（ΔZET）的程度仍未确定。 ΔZET 的值可以反映与 ET 相关的重组能或氧化还原电位，并且可以用于区分 ET 与质子耦合电子转移 (PCET)。在这里，我们合成了来自嗜热栖热菌 (truncTtRp) 的 Rieske [2Fe–2S] 亚基的蛋白质“电荷梯”，并对 ΔZETa 跨 pH 值进行了 120 次静电测量。在 pH 5-10 范围内，truncTtRp 被怀疑从 ET 转变为 PCET，然后转变为两个质子耦合 ET (2PCET)。还原后，我们发现 truncTtRp 在 pH 6.0 时变得更负一个单位（ΔZET= -1.01 ± 0.14），与单次 ET 一致； pH 8.8 时为等电点（ΔZET= -0.01 ± 0.45），与 PCET 一致；在 pH 10.6 时变得更加阳性 (ΔZET= +1.37 ± 0.60)，与 2PCET 一致。这些 ΔZET 值归因于 H154 和 H134 的质子化。在整个 pH 范围内，TtRp 的氧化还原电位（之前测量）与 H154 和 H134 的质子化能以及 truncTtRp 的 ΔZET 相关，支持 Rieske 蛋白在 Fe 配位组氨酸处的离散质子泵机制。
The degree to which redox-driven proton pumps regulate net charge during electron transfer (ΔZET) remains undetermined due to difficulties in measuring the net charge of solvated proteins. Values of ΔZETcan reflect reorganization energies or redox potentials associated with ET and can be used to distinguish ET from proton(s)-coupled electron transfer (PCET). Here, we synthesized protein “charge ladders” of a Rieske [2Fe–2S] subunit fromThermus thermophilus(truncTtRp) and made 120 electrostatic measurements of ΔZETacross pH. Across pH 5–10, truncTtRp is suspected of transitioning from ET to PCET, and then to two proton-coupled ET (2PCET). Upon reduction, we found that truncTtRp became more negative at pH 6.0 by one unit (ΔZET= −1.01 ± 0.14), consistent with single ET; was isoelectric at pH 8.8 (ΔZET= −0.01 ± 0.45), consistent with PCET; and became more positive at pH 10.6 (ΔZET= +1.37 ± 0.60), consistent with 2PCET. These ΔZETvalues are attributed to protonation of H154 and H134. Across pH, redox potentials ofTtRp (measured previously) correlated with protonation energies of H154 and H134 and ΔZETfor truncTtRp, supporting a discrete proton pumping mechanism for Rieske proteins at the Fe-coordinating histidines.