Narrow view of photosynthesis

Fluorescence line-narrowing and resonance Raman properties of various chlorophyll molecules have been measured in organic solvents. The work sheds new light on one of life's most important biochemical processes - photosynthesis - and might one day allow scientists to take another step closer to emulating the reactions to trap solar energy.

Life on earth is rooted in photosynthesis. During this process carbon dioxide is sequestered and combined with water using the energy of sunlight to form sugars and release oxygen as a byproduct. At the molecular heart of the process is chlorophyll. Chlorophyll molecules play many complex roles in the process that are not entirely understood in every detail. In antenna proteins, chlorophyll molecules trap photons and transfer the excitation energy towards the reaction centre proteins. At this stage, they assist with charge separation and initial electron transfer step.

Nature has had millions of years to optimize the physicochemical properties of this machinery through evolution. Not least, chlorophyll is fine tuned for its lowest singlet excited-state energy and its redox potential. Scientists in France and the UK have now taken a closer look at the equilibration of excitation energy within the light-harvesting proteins of higher plants and algae, which they explain often bind more than 10 chlorophyll cofactors each. They have also looked at the excitation transfers that take place between these proteins to find which particular chlorophyll molecules have the lowest energy singlet excited state.

Alison Telfer and James Barber of Imperial College London and Andrew Pascal, Luc Bordes, and Bruno Robert of the CNRS, in Gif-sur-Yvette, France explain that resonance Raman spectroscopy is a useful tool for studying such physicochemical parameters as well as investigating how sensitive they are to local interactions. They add that this technique can provide researchers with information about the structure, conformation, and configuration of the chlorophyll molecules by direct measurement of vibrational energy levels, regardless of whether it is in the free state or bound to a protein. Specifically, it is possible to elucidate the electronic state of the central magnesium ion and to find any distortions of the macrocycle that surrounds it in the chlorophyll molecule. Moreover, the details can be obtained at a resolution well below 1 angstrom.

Of course, such studies are relatively simple when one is investigating bacterial photosynthesis where there is a small number of proteins involved and each usually binds only a single pigment molecule. The study becomes much more complicated when higher plants are the subject and standard approaches to resonance Raman spectroscopy cannot yield useful results. Distinguishing between the chlorophyll molecules is not possible on the sole basis of differences in their absorptions.

Over the last three decades or so fluorescence line-narrowing (FLN) spectroscopy has yielded some pioneering information about chlorophyll because it can home in on only those molecules involved in emission and specifically that all-important lowest energy first singlet excited state.

The researchers have now used FLN to study three isolated forms of chlorophyll: a, b, and d in the organic solvent tetrahydrofuran. This solvent provides two axial ligands to the central magnesium of these molecules, something that is usually not easy to obtain for chlorophyll with only one axial ligand because the narrowing phenomenon must then be observed at very low temperatures. The team has confirmed the results with the resonance Raman spectra and used this comparison to assign specific modes for the various chlorophyll molecules.

"By observing the effects on the spectra of using different solvents we also describe the influence of molecular conformation on these band frequencies (through the coordination state of the central magnesium)," say the researchers. "These attributions should prove fundamental to the application of these vibrational techniques to the investigation of chlorophyll functions in biological materials."

"After photon absorption and/or excitation transfer, the excitation energy is rapidly equilibrated on the chlorophylls that possess the lower energy excited state. From these, the excitation energy will be re-emitted (as fluorescence) or further transferred to another protein, on the way to the photosystems," Robert told SpectroscopyNOW, "The description of these molecules is essential for our understanding of the efficiency of the light-harvesting process."

He adds that, "What FLN will bring us (and we have got already some results) is an accurate, selective, description of the chlorophyll molecules on which the excitation ultimately resides in these complex proteins which bind sometimes more than ten of these molecules." Previously, theoretical work using modified Redfield theory, for instance, has attempted to describe these chlorophylls. "With FLN, we can test the accuracy of these theories, and address the molecular parameters which underlie their properties," Robert says, "We have seen, as a first example, that in the inner antenna from the photosystem II, after equilibration, the excitation resides on one chlorophyll only, and that this chlorophyll is very likely to be highly distorted by its protein binding site."