Photosynthetic water splitting is one of the most unique biological reactions in Nature in which the visible energy of the sun provides the driving force. In the quest to find clean, non-polluting energy sources to support human society in the future, we need to learn and take advantage of the natural system.

Photosystem II (PSII) water oxidation is the main source of oxygen on earth. This most oxidizing chemistry in all of biology (>1 volt oxidation) has evolved over billions of years catalyzes rapid oxidation (>1000 turnovers. s^-1) with low thermodynamic barriers, and minimizes the generation of toxic reactive oxygen intermediates. To split water into O₂, protons and electrons, large thermodynamic barriers have to be surmounted, making this one of the most difficult chemical reactions to take place in Nature. The Gibbs free energy is at least 3.2 eV (~ 135 kJ mole^-1). In contemporary oxygenic photosynthesis the large thermodynamic barriers to water splitting are overcome by the unique properties of the PSII chlorophyll/protein complex. Remarkably, PSII has not changed significantly over the 2.2-2.5 billion years since when it first evolved, yet it is currently responsible for the annual release of ~10¹¹ tons of O₂ into the atmosphere and is essential for all aerobic life on Earth.

Bessel Kok (1) provided a major insight into the mechanism of water splitting by PSII with his finding that the reaction cycled through five intermediate states upon excitation with single turnover light flashes. The final S₄ state, being metastable, regenerates the S₀ state upon releasing O₂ (1). Detailed spectroscopic EPR and EXAFS studies have revealed that the S-state cycle and the actual water spitting chemistry occurs at a Mn₄Ca inorganic core, which undergoes redox cycling and accumulates the oxidizing equivalents generated by the PSII photochemical reaction centre (2). More recently the static structure of the PSII complex at atomic resolution is being deciphered from X-ray crystallographic analysis, and has provided the location and orientation of the Mn4Ca core and other essential cofactors with respect to the arrangement of the protein backbone (3). Nevertheless, to completely understand the water splitting reaction and to develop bio-mimetic systems for biotechnological purposes, the dynamics of the reaction have to be known. Two powerful tools to address this aspect are mass spectrometric isotope exchange measurements of the substrate water at the catalytic binding site (4) and vibrational FTIR spectroscopic measurements of chemical bond changes at the Mn₄Ca core and the associated protein ligands (5).

(1) Kok, B., Forbush, B., and McGloin, M. (1970) Photochem. Photobiol. 11, 457-475.
(2) Hillier, W., and Messinger, J. (2005) The Water: Plastoquinone Oxidoreductase in Photosynthesis (Wydrzynski, T., and Satoh, K., Eds.) pp 567-608, Springer, Printed in The Netherlands.
(3) Ferreira, K. N., Iverson, T., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838.
(4) Hillier, W., and Wydrzynski, T. (2004) Phys. Chem. Chem. Phys. 6, 4882-4889.
(5) Debus, R. J., Strickler, M. A., Walker, L. M., and Hillier, W. (2005) Biochemistry 44, 1367-1374.
Warwick Hiller home page

Scanning electron microscopy (left panel) and immunological localization of Rubisco (right panel) in a photosynthetic cell of the single cell C4 species Suaeda aralocaspica showing the Rubisco-containing chloroplasts (orange) are restricted to the proximal end of cell (right panel). (Images by Elena Voznesenskaya and Vince Franceschi)

Until 2001, the paradigm for C4 photosynthesis in terrestrial plants was the requirement for Kranz anatomy, where photosynthesis is accomplished by cooperative function of mesophyll and bundle sheath cells (1). C4 plants evolved multiple times from C3 plants, as they adapted to diverse environmental conditions causing CO2 to be limiting for photosynthesis; they exhibit amazing diversity in the structural forms of Kranz anatomy and types of C4 cycles.

The surprising discovery that C4 photosynthesis can be accomplished within a single photosynthetic cell with dimorphic chloroplasts (2-4) has broadened our view of the evolution of photosynthesis and developmental biology. The three species found to date are in genera Bienertia and Suaeda of family Chenopodiaceae. Found in central Asia and the Middle East, they grow in semi-arid and saline conditions under temperature extremes. Especially fascinating is that, between the three species, there are two novel means of spatially separating the C4 functions and dimorphic chloroplasts within the photosynthetic cells. Most of the world's major crop plants perform C3 photosynthesis. Considering global warming and increased farming of marginal land, there is interest in engineering of some crop plants to perform C4 photosynthesis. There is even evidence that C3 plants have isoforms of C4 genes, but they lack the proper expression and leaf structure to perform C4. The International Rice Research Institute, The Philippines, is forming a consortium of international scientists to improve photosynthesis in rice with the goal of genetically modifying it to perform C4 photosynthesis (5).