Stability of the S₃ and S₂ State Intermediates in Photosystem II Directly Probed by EPR Spectroscopy

The stability of the S₃ and S₂ states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor P<i>p</i>BQ at 1, 10, and 20 °C. The decay of the S₃ state was followed in samples exposed to two flashes by measuring the split S₃ EPR signal induced by near-infrared illumination at 5 K. The decay of the S₂ state was followed in samples exposed to one flash by measuring the S₂ state multiline EPR signal. During the decay of the S₃ state, the S₂ state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S₃ state to the S₁ state occurs via the S₂ state. The decay of the S₃ state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10–20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 °C) to ∼100 s (at 20 °C) in the remaining 80–90% of the centers. The decay of the S₂ state was also biphasic and showed quite similar kinetics to the decay of the S₃ state. Our experiments show that the auxiliary electron donor Y_D was oxidized during the entire experiment. Thus, the reduced form of Y_D does not participate to the fast decay of the S₂ and S₃ states we describe here. Instead, we suggest that the decay of the S₃ and S₂ states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y_Z according to S₃Y_Z ⇔ S₂Y_Z^• in the case of the S₃ state decay and S₂Y_Z ⇔ S₁Y_Z^• in the case of the S₂ state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S₃ and S₂ states occurs in PSII centers where Y_Z is also a fast donor to P₆₈₀⁺ working in the nanosecond time regime and that the fast decay of the S₃ and S₂ states occurs in centers where Y_Z reduces P₆₈₀⁺ with slower microsecond kinetics. Our measurements also demonstrate that the split S₃ EPR signal can be used as a direct probe to the S₃ state and that it can provide important information about the redox properties of the S₃ state

Probing the Lysine Proximal Microenvironments within Membrane Protein Complexes by Active Dimethyl Labeling and Mass Spectrometry

Positively charged lysines are crucial to maintaining the native structures of proteins and protein complexes by forming hydrogen bonds and electrostatic interactions with their proximal amino acid residues. However, it is still a challenge to develop an efficient method for probing the active proximal microenvironments of lysines without changing their biochemical/physical properties. Herein, we developed an active covalent labeling strategy combined with mass spectrometry to systematically probe the lysine proximal microenvironments within membrane protein complexes (∼700 kDa) with high throughput. Our labeling strategy has the advantages of high labeling efficiency and stability, preservation of the active charge states, as well as biological activity of the labeled proteins. In total, 121 lysines with different labeling levels were obtained for the photosystem II complexes from cyanobacteria, red algae, and spinach and provided important insights for understanding the conserved and nonconserved local structures of PSII complexes among evolutionarily divergent species that perform photosynthesis

L‑Edge X‑ray Absorption Spectroscopy of Dilute Systems Relevant to Metalloproteins Using an X‑ray Free-Electron Laser

L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming O K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples