Image Courtesy of Ann-Marie Abunyewa.
Water, sunlight, and a spoonful of sugar: a simple recipe that sustains much of life on Earth. Plants and other organisms famously use photosynthesis to convert light into the chemical energy that drives their lives. Central to this process is a protein complex called photosystem II (PSII), an enzyme that captures photons of light and breaks down water to release oxygen and protons.
Scientists have been interested in PSII since its discovery in the 1960s. Its relevance stems from the applicability of principles learned from biological solar fuel production to many fields, including synthetic photocatalysis, crop optimization, as well as evolutionary biology. Professor of Chemistry and Director of the Energy Sciences Institute Gary Brudvig has been studying photosynthesis for well over forty years. “His group has in many ways pioneered a lot of our most basic understanding of photosystem II,” said Christopher Gisriel, a current postdoctoral associate at the Brudvig lab.
By the 1980s, researchers had identified a photosynthetic cyanobacterium that they could easily genetically modify. Studying this species, Synechocystis sp. PCC 6803 (Syn.6803), provided useful insights into PSII’s photosynthetic mechanism, such as the specifics of water oxidation and electron transfer.
What scientists could not do, however, was solve the molecular structure of this species’ PSII protein. For biological proteins and enzymes, function is considerably affected by three-dimensional structure, like how a house-key works because of the proper arrangement of its grooves and how they fit within the lock. Because water oxidation is highly complicated, the lack of high-resolution structures to guide functional investigation has meant that many aspects of it remain unclear. “So basically, we’ve been going in somewhat blind,” Brudvig explained. “If you try to do structure-function studies with no structure, you’re kind of on thin ice.”
In an effort to fill in this gap, at the beginning of 2022, Gisriel and Brudvig’s team published a Proceedings of the National Academy of Sciences (PNAS) study reporting the first cryo-EM structure of PSII from Syn. 6803. To the authors’ surprise, there were a number of differences in the PSII structure compared to its previously presumed architecture, underscoring the need to re-examine previous data using this new structural blueprint. Not only do their findings challenge several time-honored notions about PSII’s mechanism of action, but the reported structure also provides a basis for introducing tiny changes in the protein to unlock the mysteries of biological photocatalysis.
The Troubled Heart of Photosynthesis
Much of our knowledge of PSII can be attributed to site-directed mutagenesis experiments – in which targeted changes are made to DNA – conducted in the last fifty years. In these specific experiments, scientists introduced mutations in the PSII gene to assess the role of individual amino acids, which comprise proteins, in the enzyme’s function. These studies have almost entirely been performed using Syn. 6803 cyanobacteria, which can survive with altered PSII if supplemented with glucose. This makes it an ideal model organism for mutagenesis because in many other species, mutations in PSII often led to cell death, leaving researchers unable to investigate function further.
However, the molecular structure of PSII in Syn. 6803 had remained unsolved because the organism is sensitive to the harsh conditions required for techniques like X-ray crystallography, which is used to elucidate molecular structures. To this day, the only reported structures for PSII have come from thermophilic cyanobacteria, organisms that thrive in high temperatures. However, they are poor model organisms for mutagenesis experiments due to their intolerance of growing with altered PSII.
“All this work has been going on in parallel– mutagenesis in organisms with no known structures, and structural determination in thermophiles that could not be mutated,” Brudvig said. “People just assumed that they were all the same and that they could use the thermophile as a basis for structure.” Scientists have therefore been forced to proceed with this assumption to interpret their functional data.
But this approach may not be truly justified. Firstly, there are obvious differences in the DNA sequences of the PSII genes from mesophilic and thermophilic organisms, which implies diverging structure and function. Moreover, membrane proteins from mesophilic and thermophilic organisms are generally known to have different molecular characteristics. Thus, the study of PSII function is greatly limited by the lack of a high-resolution structure for the model organism from which most biophysical data comes: Syn. 6803.
A Structural Blueprint
Large, often unstable, protein structures like PSII from Syn. 6803 are difficult, if not downright impossible, to crystallize for use in X-ray crystallography experiments. But there is now an alternative technique to visualize this three-dimensional structure: cryo-EM. Single-particle cryo-EM bombards a thin sheet of a protein solution with electrons, using a camera to detect how electron waves interact with the sample. A computer then reconstructs a 3D model of the protein from hundreds of thousands of 2D images in different orientations. “I like to think of myself as a very, very high-resolution photographer,” Gisriel said.
The Brudvig lab reported the structure of PSII from Syn. 6803 with single-particle cryo-EM at a resolution of 1.93 Angstroms (Å). For reference, the average resolution for published cryo-EM membrane protein structures is ~5Å. At this unprecedented resolution level, the Brudvig group could even see the presence of some individual protons within the complex.
PSII is biologically found in a dimeric state, with two identical monomers, each containing twenty one subunits. The core consists of four subunits, with thirteen peripheral subunits embedded in the membrane and four “extrinsic” subunits found on the inner surface of the membrane. With their novel structure in hand, the Brudvig group could now identify any major differences between the thermophilic and Syn. 6803 PSII enzymes.
Cofactors are non-proteinous molecules within an enzyme that promote its catalytic activity. Most cofactors are indeed conserved between the two species, except for a pigment called BCR101, which helps absorb light energy. Previous studies had suggested that BCR101 was important to allow PSII to dimerize, where two identical PSII proteins chemically associate. However, even without BCR101, Syn. 6803 still retains a dimeric configuration, implying that BCR101 is not as crucial for this role. Interestingly, some peripheral and extrinsic subunits, namely PsbO, PsbU, and PsbV, are quite dissimilar between PSII from the different species. This was unexpected because these subunits surround the intricately controlled “active site” of PSII, where the enzyme’s catalytic activity occurs and performs key functions in water oxidation.
The last remaining extrinsic subunit, PsbQ, is found in both thermophilic and Syn. 6803 PSII. Notably, however, PsbQ had never before been observed bound in complex with the PSII protein. Its analysis revealed that its binding in Syn. 6803 is primarily driven by unique electrostatic interactions that are not present in the thermophilic cyanobacteria. PsbQ-binding does not induce any conformational changes in the PSII complex, so the authors believe that it mainly serves to provide additional protection for the active site.
The authors were surprised to observe poor conservation of PsbO, PsbU, and PsbV between Syn. 6803 and thermophilic PSII structures. For decades, these extrinsic subunits have been thought to form channels into the active site to provide it with water to oxidize and routes for the protons and oxygen byproducts to exit. The striking differences observed in extrinsic subunit structures suggest that differences in these water channel functions are central to PSII’s enzymatic activity.
Scientists had previously identified what they considered to be three main water channels: the large, broad, and narrow channels. Although the broad and narrow channel structures are relatively well conserved between Syn. 6803 and thermophilic PSII, the most notable differences were observed in the large channel. Analysis of thermophilic structures had suggested that the large channel may play an important role in transporting water and protons to and from the active site. However, the authors found that, in Syn. 6803, the large channel is completely blocked by extension of the PsbV subunit.
Blockage of the large channel suggests that it may not actually be as crucial to PSII function as researchers had previously suggested. In fact, it is not much of a channel at all if one end appears to be closed off. These findings suggest that the narrow and broad channels may be the only ones that matter for water oxidation, which is supported by both their conservation in all known PSII structures and previous mutagenesis studies. Another plausible explanation is that PsbV may be involved in a sort of gating mechanism that selectively opens/closes the large channel.
Whichever the case, this remarkable difference between the Syn. 6803 PSII and thermophilic PSII enzymes highlights the importance of the authors’ reported structure. Without structural data from the model organism used for studying PSII, it is difficult to accurately interpret functional data, which could lead to assigning function in a manner inconsistent with true biophysical constraints.
Significance and Future Directions
Photosynthesis fuels the life of many organisms, from trees in the Arctic to hot springs cyanobacteria, to the grass outside Sterling Memorial Library. PSII is considered the only global solar fuel catalyst shared between all photosynthetic organisms and the central water oxidation enzyme. With this in mind, this research can help create a new generation of synthetic fuel catalysts, which could artificially reproduce this process of water-splitting to generate energy.
The structural differences in PSII from Syn. 6803 and thermophilic cyanobacteria have important implications in understanding the mechanism of water oxidation, suggesting that many of the field’s prior findings may now require re-examination.
With cryo-EM, researchers can observe the structure of the mutated enzyme. This work holds vast promise in unlocking the mysteries that persist in understanding the biomolecular mechanisms of photosynthesis.
Hussein, R., Ibrahim, M., Bhowmick, A., Simon, P. S., Chatterjee, R., Lassalle, L., Doyle, M., Bogacz, I., Kim, I. S., Cheah, M. H., Gul, S., de Lichtenberg, C., Chernev, P., Pham, C. C., Young, I. D., Carbajo, S., Fuller, F. D., Alonso-Mori, R., Batyuk, A., . . . Yano, J. (2021). Structural dynamics in the water and proton channels of photosystem II during the S2 to S3 transition. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-26781-z
Gisriel, C. J., Wang, J., Liu, J., Flesher, D. A., Reiss, K. M., Huang, H. L., Yang, K. R., Armstrong, W. H., Gunner, M. R., Batista, V. S., Debus, R. J., & Brudvig, G. W. (2021). High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803. Proceedings of the National Academy of Sciences, 119(1), e2116765118. https://doi.org/10.1073/pnas.2116765118