Optical spectroscopy of the red forms of Photosystem II and I

Abstract

Research in photosynthesis encompasses a wide array of complex and interconnected processes, requiring the incorporation of concepts from physics, chemistry and biology.

This thesis contributes to the understanding of photosynthesis by using low temperature optical spectroscopy to study Photosystem II (PSII) and Photosystem I (PSI). These two catalytic protein complexes are responsible for splitting water to provide electrons for the photosynthetic process, and transferring these electrons across the thylakoid membrane, respectively

These enzyme complexes provide a particular challenge for optical spectroscopy as they contain large numbers of chromophores with overlapping absorption bands, which require a combination of optical techniques to separate out. In this work, the techniques of absorption, fluorescence, Circular Dichroism (CD), Magnetic Circular Dichroism and electrochromism have been used to study these enzymes.

This thesis has two main themes: the direct observation of the optical spectroscopic properties of the Oxygen Evolving Complex (OEC) in Photosystem II (Publications 1 and 4) and the location of far-red absorbing chlorophylls in PSII and PSI of a red-light adapted cyanobacterium (Publication 2 and 3)

The OEC of PSII is the tetramanganese cluster where electrons are extracted in a stepwise fashion from water, resulting in the release of oxygen. In this process, the cluster passes through four metastable S-states. In Publication 1, variable temperature and variable field (VTVH) MCD is used as a tool to identify the spectrum of each S-state in an optically transparent window in the region of 720-800nm.

Low temperature MCD spectra of this region shows a series of sharp peaks that are S-state dependent. These features were assigned to a series of Mn(IV) 4A -> 2E "spin flip" transitions. These spectra and their associated assignments provide a new perspective on the electronic and magnetic structure of the OEC.

Publication 4 follows on from 1, focusing on the two sub-forms of the S2 state of the OEC. A combination of EPR and MCD enables the identification of these two states as being redox isomers and give insight into the mechanisms of water oxidation.

Chroocooccidiopsis thermalis is an extremophile cyanobacteria that can grow in hot and dry conditions. When grown under red-light conditions, this cyanobacteria can produce red absorbing chlorophylls d and f, along with chlorophyll a.

In Publication 2, using a range of biophysical techniques, including low temperature optical spectroscopy, we clearly establish that these red absorbing chlorophylls pla a key role in both PSI and PSII and are not just as long wavelength light harvesting pigments as previously reported. Specifically, charge separation in PSI uses chlorophyll f at 745nm and PSII uses chlorophyll d or f at ~727nm. The organism is shown to be able to perform photosynthesis with lower energy light than previously thought possible.

Publication 3 is an extension of Publication 2 where the location and function of the red absorbing chlorophyll at ~727nm of PSII in C.thermalis is further investigated. Modelling of low temperature spectroscopic data, CD and electrochromism in particular, yields more information about the location of this pigment within the PSII complex. Previously, ChlD1 was assigned as the most likely candidate for the primary donor. However, the analysis in Publication 3 favours PD2 as the primary donor. This unexpected result requires further experimentation and calculational studies to verify.

This thesis shows the strength of applying multiple spectroscopy techniques to complex biological systems and how such data can be used to extract information about both the structure and function of these proteins.