Time-Resolved Single Frequency Infrared Absorption Spectroscopy on Photosystem II

In spite of recent advancements in structure elucidation of Photosystem II (PSII), by far not the complete catalytic cycle of photosynthetic water oxidation is sufficiently well understood. Open questions concern the type of O-O bond formation mechanism, the nature of the substrate water molecules, the role of protein-internal H-bond networks and the sites of proton release and exit. Time-resolved infrared (IR) spectroscopy provides information about protonation states, H-bonding networks and bond strengths. On oxygen-evolving PSII, there have been less than five reports of time-resolved IR spectroscopy, but either they presented only a few wavenumbers of pure spectral resolution (16 cm-1) or were not free of distortion by a laser-induced heat artefact. In this thesis, a new alternative experiment is described that employs a continuous-wave Quantum-Cascade-Laser for single-frequency IR (SFIR) detection of absorption transients induced by a series of nanosecond laser flashes (532 nm). It became possible to detect IR absorption smaller than 10−5 OD in the time domain from 40 ns to s at wavenumbers ranging from 1300 to 1650 cm-1. The setup comprises automatic sample exchange, data recording, a temperature control system and an adapted measurement protocols. The latter includes a flash sequence that drives the PSII synchronously through its reaction cycle (the S-state cycle PSII) and allows to correct for the flash-induced heat artefact, reproducibly. Reliable data now can be recorded at ten wavenumbers per day. (1) First, FTIR steady-state measurements, variable chlorophyll fluorescence and the SFIR experiment were combined to track the electron transfer from the first (QA) to the second (QB) quinone electron-acceptor of PSII. The decay kinetics of Q − A were found to be identical in the SFIR transients at 1478 cm-1 and the variable fluorescence yield. This finding and the analysis of the ”connectivity” resulting from inter-PSII excitation energy transfer revealed that connectivity-corrected fluorescence traces the QA-QB electron transfer very well, thereby verifying assumptions and models used thousands of times in physiological studies. (2) I contributed to an ongoing discussion about the position of the CO stretching vibration of the redox active tyrosine radical Y•ox Z . Using the high spectral resolution, a clear signal assignable to Y•ox Z could be identified at 1512.5-1515 cm -1. The band formed faster than 40 ns and decayed with the known Mn oxidation kinetics. Its position is the same as in Mn depleted samples, surprisingly suggesting similar H-bond properties. (3) I could show that the symmetric stretching region of carboxylates senses almost all kinetic components of the previously proposed extended reaction cycle of PSII water oxidation, which is characterized by alternating removal of electrons and protons from the catalytic site of water oxidation. Moreover, three so far unidentified bands showing either a 300 ns or a 30 μs phase are found in the S2 → S3 and S3 → S0 transitions. (4) With Richard Debus (UC Riverside), we established a protocol to investigate PSII core complexes extracted from Synechocystis with time-resolved SFIR. First, it was verified that the kinetics of the reaction cycle of the cyanobacterial PSII are identical to that of spinach PSII. Then I obtained the very first time-resolved IR transients on genetically modified PSII. Introducing an alanine instead of an aspartate at the crucial 61 position of the D1 protein did not slow down the fast proton release phase in S2 → S3 and S3 → S0, but the Mn oxidation – especially in S3 → S0. This excludes a direct involvement of the D61 residue in the early proton release but creates new questions on the mechanism of water oxidation. In summary, the development of a new (superior) experimental approach for time-resolved tracking of the events in the PSII reaction cycle has facilitated new insights and opens up a variety of new avenues in PSII research.