Department of Chemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
Biochemistry. 2013 Jul 16;52(28):4758-73. doi: 10.1021/bi301700u. Epub 2013 Jul 3.
The role of chloride in photosystem II (PSII) is unclear. Using structural information from PSII and a careful comparison with other chloride-activated enzymes, we proposed a role for chloride at the D2-K317 site in PSII [Pokhrel, R., et al. (2011) Biochemistry 50, 2725-2734]. To probe the role of chloride at this site, the D2-K317R, D2-K317A, D2-K317Q, and D2-K317E mutations were created in the cyanobacterium Synechocystis sp. PCC 6803. Purified PSII from the mutants was probed with Fourier transform infrared difference spectroscopy, demonstrating that compared to PSII from wild-type Synechocystis, PSII from all four mutants exhibit changes in the conformations of the polypeptide backbone and carboxylate groups. However, D2-K317R PSII exhibits minor changes, whereas D2-K317A, D2-K317Q, and D2-K317E PSII exhibit more substantial changes in polypeptide conformations. Steady-state oxygen-evolution measurements of purified PSII core complexes show that the oxygen-evolution activity of D2-K317A is independent of chloride. This is consistent with the loss of the chloride requirement when the charged K residue is replaced with an uncharged residue that no longer binds to an essential carboxylate (D1-D61) in the absence of chloride, analogous to observations in other chloride-activated enzymes. In contrast, the oxygen-evolution activity of D2-K317R is sensitive to the chloride concentration in the assay buffer; the effective KD for chloride binding is higher in D2-K317R than in wild-type PSII, possibly because of a less optimal binding site in the mutant. The S2 states of wild-type, D2-K317A, and D2-K317R PSII were probed using electron paramagnetic resonance spectroscopy. A g = 2 multiline signal, similar to the wild-type signal, was observed for D2-K317A and D2-K317R. However, a g = 4 signal was also observed for D2-K317R. Measurements of flash-dependent O2 yields showed that D2-K317A and D2-K317R have a higher miss factor than wild-type PSII. The oxygen-release kinetics of D2-K317A and D2-K317R were slower than those of the wild type, in the following order: D2-K317A < D2-K317R < wild type. These results collectively suggest that proton transfer is inefficient in D2-K317A and D2-K317R, thereby giving rise to a higher miss factor and slower oxygen-release kinetics.
氯离子在光系统 II（PSII）中的作用尚不清楚。我们利用 PSII 的结构信息，并与其他氯离子激活酶进行仔细比较，提出了氯离子在 PSII 的 D2-K317 位点的作用[Pokhrel, R., 等人。（2011）生物化学 50, 2725-2734]。为了探究该位点氯离子的作用，我们在蓝藻集胞藻 PCC 6803 中创建了 D2-K317R、D2-K317A、D2-K317Q 和 D2-K317E 突变体。用傅里叶变换红外差谱法探测突变体的 PSII，结果表明，与野生型集胞藻的 PSII 相比，所有四种突变体的 PSII 的多肽骨架和羧酸盐基团的构象都发生了变化。然而，D2-K317R PSII 的变化较小，而 D2-K317A、D2-K317Q 和 D2-K317E PSII 的多肽构象变化较大。纯化 PSII 核心复合物的稳态氧释放测量表明，D2-K317A 的氧释放活性不依赖于氯离子。这与在没有氯离子的情况下，带电荷的 K 残基被不带电荷的残基取代时，当不带电荷的残基不再与氯离子激活酶中必需的羧酸盐（D1-D61）结合时，氯离子的需求消失是一致的。相比之下，D2-K317R 的氧释放活性对测定缓冲液中的氯离子浓度敏感；D2-K317R 的氯离子结合的有效 KD 高于野生型 PSII，这可能是由于突变体中结合位点不太理想。用电子顺磁共振波谱法探测了野生型、D2-K317A 和 D2-K317R PSII 的 S2 态。对于 D2-K317A 和 D2-K317R，观察到类似于野生型信号的 g = 2 多线信号。然而，对于 D2-K317R，也观察到了 g = 4 信号。闪光依赖性 O2 产率的测量表明，D2-K317A 和 D2-K317R 的漏失因子高于野生型 PSII。D2-K317A 和 D2-K317R 的氧气释放动力学比野生型慢，顺序为：D2-K317A < D2-K317R < 野生型。这些结果共同表明，在 D2-K317A 和 D2-K317R 中质子转移效率较低，从而导致漏失因子较高和氧气释放动力学较慢。
