Visualization of CryoEM Hydrogen Peaks in the Photosystem II Reaction Center P 680 Chlorophyll Pair

Abstract X-ray crystallography reports only on the electron density (ED) distribution in molecules, and neutron crystallography only describes the distribution of nuclei. CryoEM visualizes both simultaneously and thus can provide unique information about the electronic structures of molecules that neither of its competitors can do alone. Hydrogen peaks in CryoEM maps are an order of magnitude larger than those in X-ray crystallographic maps. This makes it possible to visualize them in CryoEM maps even when they are not fully resolved from the peaks of the heavy atoms to which they are bonded. Here, I report the results obtained when the methods I have developed for locating the hydrogen peaks were applied to photosystem II (PSII) reaction center P680 chlorophyll pair (A404/D404), its protein ligands (D1-H198 and D2-H197), and flanking residues (I192-G204 of the D1 subunit and T191-A202 of D2) in a CryoEM map having a resolution of 1.71 Å. Hydrogen peaks were found at all the chemically expected locations except for two. First, the orientations of the two methylene hydrogen peaks of the vinyl substituent on A404 were found to differ from those of the same vinyl methylene hydrogens of all the other chlorophylls in PSII. They were orthogonal to the expected plane instead of being in-plane. Second, in the backbone I192O–H195N hydrogen-bonding (HB) pair, the hydrogen peak was found to be next to D1-I192O, rather than D1-H195N, which indicates that this hydrogen atom is part of a carboxyl-imide hydrogen bond rather than a carbonyl-amide hydrogen bond.