We report a structure-based biological approach to identify the proton-transfer pathway in photosystem II. First, molecular dynamics (MD) simulations were conducted to analyze the H-bond network that may serve as a Grotthuss-like proton conduit. MD simulations show that D1-Asp61, the H-bond acceptor of H₂O at the Mn₄CaO₅ cluster (W1), forms an H-bond via one water molecule with D1-Glu65 but not with D2-Glu312. Then, D1-Asp61, D1-Glu65, D2-Glu312, and the adjacent residues, D1-Arg334, D2-Glu302, and D2-Glu323, were thoroughly mutated to the other 19 residues, i.e., 114 Chlamydomonas chloroplast mutant cells were generated. Mutation of D1-Asp61 was most crucial. Only the D61E and D61C cells grew photoautotrophically and exhibit O₂-evolving activity. Mutations of D2-Glu312 were less crucial to photosynthetic growth than mutations of D1-Glu65. Quantum mechanical/molecular mechanical calculations indicated that in the PSII crystal structure, the proton is predominantly localized at D1-Glu65 along the H-bond with D2-Glu312, i.e., pK_a(D1-Glu65) > pK_a(D2-Glu312). The potential-energy profile shows that the release of the proton from D1-Glu65 leads to the formation of the two short H-bonds between D1-Asp61 and D1-Glu65, which facilitates downhill proton transfer along the Grotthuss-like proton conduit in the S₂ to S₃ transition. It seems possible that D1-Glu65 is involved in the dominant pathway that proceeds from W1 via D1-Asp61 toward the thylakoid lumen, whereas D2-Glu312 and D1-Arg334 may be involved in alternative pathways in some mutants.

我们报告了一种基于结构的生物学方法来识别光系统II中的质子转移途径。首先，进行了分子动力学（MD）模拟，以分析可能用作格罗特斯式质子导管的氢键网络。MD模拟显示，D1-Asp61是Mn ₄ CaO ₅簇（W1）上H ₂ O的H键受体，它通过一个水分子与D1-Glu65而不是D2-Glu312形成H键。然后，将D1-Asp61，D1-Glu65，D2-Glu312和相邻残基D1-Arg334，D2-Glu302和D2-Glu323彻底突变为其他19个残基，即产生了114个衣藻衣藻叶绿体突变体细胞。 。D1-Asp61的突变是最关键的。仅D61E和D61C细胞自养生长并显示O_2-不断发展的活动。D2-Glu312的突变对光合生长的重要性不如D1-Glu65的突变。量子力学/分子力学计算表明，在PSII晶体结构中，质子主要位于与D2-Glu312沿H键的D1-Glu65处，即p K _a（D1-Glu65）> p K _a（D2- Glu312）。势能图显示，质子从D1-Glu65释放会导致在D1-Asp61和D1-Glu65之间形成两个短的氢键，这有利于质子沿着Grotthuss样质子导管在质子传递过程中向下移动。 S ₂至S ₃过渡。D1-Glu65可能参与了从W1经由D1-Asp61到达类囊体腔的主要途径，而D2-Glu312和D1-Arg334可能参与了某些突变体的替代途径。