Time-Resolved Single-Frequency Infrared Spectroscopy Provides Atomistic Insight into the Functioning of the Two Photosystems of Oxygenic Photosynthesis

Abstract

Photosystems I and II (PSI, PSII) are two large protein complexes that are part of the electron transport chain of oxygenic photosynthesis in plants, algae, and cyanobacteria. The activity of these photosystems is indispensable in oxygenic photosynthesis, the process that powers life on Earth by converting solar energy into chemical energy. PSII harbors the light-driven water splitting process, which results in the removal of four protons and four electrons from two water molecules. This process is coupled to the release of molecular oxygen (O2) and is thus also referred to as photosynthetic oxygen evolution or oxygen evolution reaction (OER); it is responsible for creating the oxygen in the Earth’s atmosphere. The OER cycle of PSII (S-state cycle) involves four successive photon absorption events, which lead to the stepwise accumulation of four oxidation equivalents at the catalytic site, a protein-bound Mn4CaOx cluster, prior to O2 formation. In contrast, the PSI reactions can be discussed as a one-photon process: upon absorption, charge separation takes place and the electron is transported down one of two paths (the A- or B-branch), finally reducing the mobile electron carrier ferredoxin. In both photosystems, the light-induced processes are still insufficiently understood regarding the temporal sequence of atomistic events.

The focus of this thesis is the investigation of the S-state transitions in PSII by means of time-resolved single-frequency infrared (TRSF-IR) spectroscopy in the wavenumber range from 1800 cm–1 to 1300 cm–1. Measurements on spinach PSII membrane particles in H2O, D2O and at several different pH values allowed for the characterization of the kinetics of three S-state transitions (S1→S2, S2→S3 and S3→S0). Global analysis of time-resolved spectral data sets resulted in decay-associated spectra (DAS), which are essentially spectral fingerprints of individual kinetic phases. The DAS of the proton transfer (PT) and electron transfer (ET) phases of the O2-producing S3→S0 transition mostly reproduced the results of a previous step-scan Fourier-transform infrared (FTIR) study (Greife et al., 2023), but also managed to resolve a crucial deuteration-induced band shift, which was left undetected by the FTIR study.

Global analysis of a data set on a spinach PSII sample depleted of its Mn4CaOx cluster resulted in DAS that showed great similarity to previously reported FTIR spectra of two PSII co-factors (QA and P680). Double difference spectra of intact minus Mn depleted PSII revealed that certain spectral regions are likely to be more strongly affected by a high fraction of broken PSII centers than others.

The comparative analysis of spinach PSII membranes to PSII core particles from Thermosynechococcus vestitus BP-1 (T. vestitus) and Synechocystis sp. PCC 6803 revealed that while at some wavenumbers the IR difference transients are very similar to each other, they differ drastically at others. It was found that the likely source of this observation is a differing amount of broken PSII centers in the different samples. Measurements of T. vestitus in D2O revealed similar kinetic isotope effects as observed for spinach PSII. PSII from Synechocystis carrying either the D1-N298A or the D1-D61A mutation was investigated and compared to wild-type PSII. While the monophasic O2-producing ET step of the S3→S0 transition was found to be drastically slowed in D1-D61A, it was found to be biphasic in D1-N298A. We assign the two observed kinetic phases to (i) O2-evolution occurring at a similar rate as in wildtype PSII and (ii) strongly decelerated substrate water insertion into the Mn4CaOx cluster, which in wild-type PSII occurs faster than the rate-limiting step and is therefore usually not spectroscopically detectable.

Measurements on three PSI wild-type variants allowed for the first ever IR spectroscopic observation of ET down the A- and B-branch at room temperature. Global analysis of the nanosecond kinetics resulted in DAS of forward ET down the A-branch. All in all, TRSF-IR measurements allowed for the kinetic and spectral characterization of various PSII and PSI samples, resulting in a small contribution toward a full understanding of the molecular mechanisms of oxygenic photosynthesis.