Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Nature. 2017 Mar 2;543(7643):131-135. doi: 10.1038/nature21400. Epub 2017 Feb 20.


Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis / radiation effects
  • Crystallography / methods*
  • Cyanobacteria / chemistry
  • Electron Transport / radiation effects
  • Electrons*
  • Fourier Analysis
  • Lasers*
  • Light*
  • Manganese / chemistry
  • Manganese / metabolism
  • Models, Molecular
  • Nonheme Iron Proteins / chemistry
  • Nonheme Iron Proteins / metabolism
  • Nonheme Iron Proteins / radiation effects
  • Oxygen / chemistry*
  • Oxygen / metabolism
  • Oxygen / radiation effects*
  • Photosystem II Protein Complex / chemistry*
  • Photosystem II Protein Complex / metabolism
  • Photosystem II Protein Complex / radiation effects*
  • Protons
  • Temperature
  • Time Factors
  • Water / chemistry
  • Water / metabolism


  • Nonheme Iron Proteins
  • Photosystem II Protein Complex
  • Protons
  • Water
  • Manganese
  • Oxygen