Thomas Wydrzynski

Oxygen ligand exchange at metal sites ^ implications for the O2 evolving mechanism of photosystem II

The mechanism for photosynthetic O2 evolution by photosystem II is currently a topic of intense debate. Important questions remain as to what is the nature of the binding sites for the substrate water and how does the O-O bond form. Recent measurements of the 18O exchange between the solvent water and the photogenerated O2 as a function of the S-state cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399-4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S0 state, while the second substrate water molecule binds in the S3 state or possibly earlier. It may be that the second substrate water molecule only enters the catalytic sequence following the formation of the S3 state. Most importantly, comparison of the observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water molecules are most likely bound to separate Mn(III) ions, which do not undergo metal-centered oxidations through to the S3 state. The implication of this analysis is that in the S1 state, all four Mn ions are in the +3 oxidation state. This minireview summarizes the arguments for this proposal.

Expression of the Manganese Stabilising Protein from a Primitive Cyanobacterium

The Manganese Stabilising Protein (MSP) is an extrinsic subunit of Photosystem II, which is essential for optimal levels of oxygen production during photosynthesis under physiological conditions. The MSP is present in all oxygenic phototrophs including Gloeobacter violaceus, which is the most primitive of contemporary cyanobacteria, having diverged very early from the common ancestor. Because G. violaceus is difficult to culture and is extremely slow growing, it has not yet been isolated biochemically. Instead we report here on our protocols for the expression and purification of rMSP in E. coli inclusion bodies.

The evolution of Photosystem II: insights into the past and future

This article attempts to address the molecular origin of Photosystem II (PSII), the central component in oxygenic photosynthesis. It discusses the possible evolution of the relevant cofactors needed for splitting water into molecular O2 with respect to the following functional domains in PSII: the reaction center (RC), the oxygen evolving complex (OEC), and the manganese stabilizing protein (MSP). Possible ancestral sources of the relevant cofactors are considered, as are scenarios of how these components may have been brought together to produce the intermediate steps in the evolution of PSII. Most importantly, the driving forces that maintained these intermediates for continued adaptation are considered. We then apply our understanding of the evolution of PSII to the bioengineering of a water oxidizing catalyst for utilization of solar energy.

Investigation of substrate water interactions at the high-affinity Mn site in the photosystem II oxygen-evolving complex.

18O isotope exchange measurements of photosystem II (PSII) in thylakoids from wild-type and mutant Synechocystis have been performed to investigate binding of substrate water to the high-affinity Mn4 site in the oxygen-evolving complex (OEC). The mutants investigated were D1-D170H, a mutation of a direct ligand to the Mn4 ion, and D1-D61N, a mutation in the second coordination sphere. The substrate water 18O exchange rates for D61N were found to be 0.16±0.02 s−1 and 3.03±0.32 s−1 for the slow and fast phases of exchange, respectively, compared with 0.47±0.04 s−1 and 19.7±1.3 s−1 for the wild-type. The D1-D170H rates were found to be 0.70±0.16 s−1 and 24.4±4.6 s−1 and thus are almost within the error limits for the wild-type rates. The results from the D1-D170H mutant indicate that the high-affinity Mn4 site does not directly bind to the substrate water molecule in slow exchange, but the binding of non-substrate water to this Mn ion cannot be excluded. The results from the D61N mutation show an interaction with both substrate water molecules, which could be an indication that D61 is involved in a hydrogen bonding network with the substrate water. Our results provide limitations as to where the two substrate water molecules bind in the OEC of PSII.

Engineering model proteins for Photosystem II function

Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O2 and H2. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.

Introduction to Photosystem II

This chapter briefly traces some of the early studies and key findings which have led to our current perception and understanding of Photosystem II, the water:plastoquinone oxidoreductase in oxygenic photosynthesis. Starting with the discovery of oxygen and the idea of two photosystems, the progressive identification of the unique structural and functional aspects of Photosystem II are outlined and related to the corresponding chapters in the book. The aim is to integrate the detailed descriptions in the various chapters in the context of the structure and function of Photosystem II as a whole. The chapter ends with a brief perspective for the future study and application of Photosystem II research.

Water splitting by Photosystem II—where do we go from here?

As this special issue shows, we know quite a lot about the workings of Photosystem II and the oxidation of water to molecular O2. However, there are still many questions and details that remain to be answered. In this article, I very briefly outline some aspects of Photosystem II electron transport that are crucial for the efficient oxidation of water and require further studies. To fully understand Photosystem II reactions is not only a satisfying intellectual pursuit, but is also an important goal as we develop new solar technologies for the splitting of water into pure O2 and H2 for use as a potential fuel source. “As Students of the Past, We Send Greetings to the Students of the Future.”*

Molecular solar fuels

Part I: Perspectives on Molecular Solar Fuels Chapter 1: Solar Energy Utilization Chapter 2: Engineering Low-Barrier Photocatalysts Part II: The Capture of Solar Energy Chapter 3: Bacteriorhodopsins - The Simplest Phototransducers Chapter 4: Photosynthetic Light-Harvesting Complexes - The Most Efficient Light Gatherers Chapter 5: Synthetic Light-Harvesting Pigment Arrays Part III: Photochemical Conversion of Solar Energy Chapter 6: Natural Photosynthetic Reaction Centers - Charge Separation with High Quantum Yields Chapter 7: Wired Reaction Centers Chapter 8: Bioelectrodes Chapter 9: Charge Stabilization in Polymer Films Part IV: Storage of Solar Energy Chapter 10: The Photosynthetic Water-Splitting Complex Chapter 11: Biomimics of the Water-Splitting Active Site Chapter 12: Biological H2 Generation Chapter 13: Biomimics of the hydrogenase active site Part V: Future Goals Chapter 14: Photocatalysts that Split Water and Produce H2 and O2 Within the Same Molecular Assembly Chapter 15: Light-driven water oxidation and CO2 reduction Chapter 16: Synthetic Biology

The importance of protein-protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin--manganese stabilising protein.

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.

Early Indications for Manganese Oxidation State Changes During Photosynthetic Oxygen Production: A Personal Account

One of the major questions yet to be answered in photosynthesis research today is what is the chemical mechanism for the oxidation of water into molecular oxygen. It is well established that an inorganic cluster of four manganese ions and at least one calcium ion form the catalytic core. As the oxidation potential generated by the Photosystem II reaction center is accumulated over the four sequential steps needed to produce O2, changes in the oxidation state of the catalytic manganese occur, though the formal oxidation states that are involved are still a matter of considerable debate. Much of what is currently known has come from direct measurements of the catalytic manganese using electron paramagnetic resonance (EPR) and X-ray spectroscopy. However, in the early attempts to attack this problem, the catalytic manganese was monitored indirectly by its paramagnetic effect on the nuclear magnetic resonance (NMR) relaxation rates of solvent water protons. In this contribution, a description of the proton relaxation rate phenomenon and its use to indicate manganese oxidation state changes during O2 production is presented.