Proton transfer pathway from the oxygen-evolving complex in photosystem II substantiated by extensive mutagenesis

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 O-2-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 (D-2-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.

我们报道了一种基于结构的生物学方法来确定光系统II中的质子传递途径。首先，进行了分子动力学（MD）模拟，以分析可能作为类似格罗特斯质子通道的氢键网络。MD模拟表明，D1 - 天冬氨酸61（Mn4CaO5簇（W1）处H2O的氢键受体）通过一个水分子与D1 - 谷氨酸65形成氢键，但不与D2 - 谷氨酸312形成氢键。然后，将D1 - 天冬氨酸61、D1 - 谷氨酸65、D2 - 谷氨酸312以及相邻的残基D1 - 精氨酸334、D2 - 谷氨酸302和D2 - 谷氨酸323彻底突变为其他19种残基，即产生了114个衣藻叶绿体突变细胞。D1 - 天冬氨酸61的突变最为关键。只有D61E和D61C细胞能够光合自养生长并表现出放氧活性。D2 - 谷氨酸312的突变对光合作用生长的关键程度低于D1 - 谷氨酸65的突变。量子力学/分子力学计算表明，在光系统II晶体结构中，质子主要沿着与D2 - 谷氨酸312的氢键定位于D1 - 谷氨酸65，即pK（a）（D1 - 谷氨酸65）> pK（a）（D2 - 谷氨酸312）。势能曲线表明，质子从D1 - 谷氨酸65的释放导致D1 - 天冬氨酸61和D1 - 谷氨酸65之间形成两个短氢键，这有利于在S2到S3转变过程中沿着类似格罗特斯质子通道的顺坡质子传递。似乎D1 - 谷氨酸65参与了从W1经由D1 - 天冬氨酸61通向类囊体腔的主要途径，而D2 - 谷氨酸312和D1 - 精氨酸334可能在一些突变体中参与替代途径。