de Lichtenberg Casper, Rapatskiy Leonid, Reus Michael, Heyno Eiri, Schnegg Alexander, Nowaczyk Marc M, Lubitz Wolfgang, Messinger Johannes, Cox Nicholas
Department of Chemistry- Ångström Laboratorium, Uppsala University, Uppsala S-75120, Sweden.
Department of Chemistry, Chemical Biological Centre, Umeå University, Umeå S-90187, Sweden.
Proc Natl Acad Sci U S A. 2024 Mar 12;121(11):e2319374121. doi: 10.1073/pnas.2319374121. Epub 2024 Mar 4.
Identifying the two substrate water sites of nature's water-splitting cofactor (MnCaO cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved O-Electron-electron Double resonance detected NMR (TR-O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of HO to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the MnCaO cofactor after mixing with HO. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (W) in the TR-MIMS experiments and ii) that the exchange rates of O5 and W are both enhanced by Ca→Sr substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being W. This strongly restricts options for the mechanism of water oxidation.
确定自然界水裂解辅因子（MnCaO簇）的两个底物水位点，为解析光系统II（PSII）中O-O键形成机制提供了重要信息。为此，我们采用时间分辨膜进样质谱（TR-MIMS）和时间分辨O-电子-电子双共振检测核磁共振（TR-O-EDNMR）方法，对天然Ca-PSII和生物合成取代的Sr-PSII的S态进行了平行底物水交换实验。TR-MIMS可解析在向介质中添加H₂¹⁸O后，氧同位素标记掺入底物位点的动力学过程，而磁共振技术原则上可在与H₂¹⁸O混合后，对MnCaO辅因子的所有可交换氧配体进行表征。这种独特的组合表明：i）在实验条件下，从……分离出的Ca-PSII核心复合物的中心氧桥（O5）与TR-MIMS实验中缓慢交换的底物水（W）具有相同的交换速率；ii）Ca→Sr取代以类似方式提高了O5和W的交换速率。结合之前的TR-MIMS结果，这表明只有O5满足作为W的所有标准。这极大地限制了水氧化机制的可能性。
