Remember when I told you that the light reactions worked like this?
Well, that’s not completely true.* Everything makes perfect sense in 2D. It’s easy to follow the electrons. The problem is this- life doesn’t work well compressed down to 2D in perfect one to one to one ratios. It’s much more complicated. It’s 3D. It’s greater than the sum of its parts.
Here’s a great example of that brought to you by Liu** et al from the Blankenship group at Washington University in St. Louis from the current issue of Science. They report the isolation and characterization of a megacomplex consisting of Photosystem II (PSII), Photosystem I (PSI) and the Phycobilisome (PBS). Sure, lots of people have studied these complexes independently, but we know they have to work coordinately. This is our first glimpse of how they really fit together. It is a biological example of Voltron, where working alone can only get you so far. All of the components must come together in a kick*ss megarobot megacomplex to really get the job done.
First let’s review some background, PSII uses light energy to pull electrons from water and initiate the electron transfer chain, and PSI uses light energy to transfer electrons from the carrier protein plastocyanin (PC) to Ferredoxin (Fd). While both of these photosystems have numerous chlorophyll pigments to capture light, they need a little help in the form of an antenna complex that ensures a steady stream of photon energy into each photosystem.
In cyanobacteria, the antenna complex is the phycobilisome (PBS). This is a large complex (think giant blue octopus) made up of soluble bilin pigment-containing proteins that sits on the cytoplasmic side of the thylakoid membrane. The blue bilin pigments are able to complement the range of visible light absorbed by cyanobacteria, taking advantage of the 500 – 650 nm range. Compare that to the figure that shows the absorption spectra of chlorophyll alone. It’s the job of the PBS to absorb this light energy and transfer it to PSII or PSI. In this way, neither of the photosystems will have to wait very long for their next hit of light energy.
Liu and co-authors combined a few different techniques to capture and characterize this PSII-PSI-PBS megacomplex. First, they had to design a new way to isolate PSII by creating an affinity-tagged version of the PsbO protein. This places the affinity tag on the lumenal side of the complex in contrast to the typical PSII isolation, involving a tagged CP47 protein (tag on the cytoplasmic side and may be hidden by a giant PBS on the same side). Next, they used a mild crosslinking treatment to covalently link together proteins that were physically close to one another in the cells. When they performed the purification procedure on the crosslinked material, they identified the proteins present. They specifically identified the PSII, PSI and PBS proteins as part of a megacomplex. The key is in the chemical crosslinkers that glue the pieces together such that they survive biochemical purification. This kind of megacomplex is held together in the cells with a multitude of non-covalent interactions and does not survive standard biochemical preparation procedures (they are just too harsh, no matter how fast and cold they are performed).
Mapping the position of the chemical crosslinkers on specific peptides using mass spectrometry enabled them to precisely identify which residues of the different proteins were connected with one another. Combining that information with the individual structures of each of the complexes (PSII, PSI and PBS), they were able to reconstruct a 3D picture of how they come together in a megacomplex. It looks like this***:
The PBS contains the allophycocyanin core (shown in yellow and green) with only a few individual discs of phycocyanin rods (blue). These rods could extend further (i.e. the arms of the blue octopus). Beneath the PBS is a PSII (dimer) and two PSI (trimer) complexes. This should give you a better perspective on scale for each of these complexes.
Liu and coauthors were also able to perform time-resolved fluorescence spectroscopy to confirm that there is energy transfer from the PBS to each of the photosystems in the isolated megacomplex. This provides evidence that you’re looking at something biologically relevant and not just random pieces that got glued together by the crosslinkers. You can also take a closer look at the details of how the pieces specifically fit together (crosslinker mapping data), which confirms that this megacomplex is likely a form found in the cell.
In the end, this makes a lovely picture, but it raises a ton of new questions. The first among them is how the PBS ‘decides’ which photosystem (I vs. II) gets energy when. We’ve known for a long while that the cells are able to control energy balance between the two photosystems according to the needs of the cells. This means that PSII and PSI are not working/receiving excitation energy equally. The cells have mechanisms for preferentially ‘feeding’ light energy to one over the other for optimal electron flow. Some hypotheses had the PBS moving on top of the membrane to center themselves over either PSI or PSII, but the sheer size and this new information calls that into question.
Here’s another link to the reference: http://www.sciencemag.org/content/342/6162/1104.full
*I’m not conceding that I lied to you. It’s just “You can’t handle the truth!”
**Shout out for photosynthesis research in a glam journal by a Bricker lab alum! Haijun was a graduate student here in the Bricker lab and we overlapped here at LSU for some time. He moved onto a postdoc in the Pakrasi lab (note: there’s no official exchange program) where he took over some of my leftover projects. He applied the same protein complex purification with cross-linker mapping techniques to further understand PSII assembly. He is currently in the Blankenship lab continuing his research on photosystems and their antenna complexes.
***This figure isn’t in the paper, but was generated as potential cover art by Haijun Liu (labels added by me for clarity here on this blog). Beautiful, no? Well, this was not chosen as the Science cover. Instead a picture of rust (iron oxide) was used- WTF?