An X-ray of smoke bush leaves, highlighting the branching veins throughout the leaves.

Photosynthesis is still a mystery—but science is revealing unknown steps

Most plants split water molecules to generate energy, and now we have a clearer picture of exactly how they do it.

Researches have found missing steps in photosynthesis by stimulating plant proteins with laser light and then capturing the resulting processes with x-rays. Here, the leaves of a smoke bush are visualized via x-ray.

Photosynthesis is a vital part of life on Earth, providing plants with the means to make their own food—but scientists still don’t know exactly how it works. Two new experiments have revealed a missing step in photosynthesis, identifying previously unknown details of how water molecules are broken apart, one of the most difficult reactions.

When the water molecules are split, oxygen is released into the air. That oxygen, “which we all rely on and is essential for all higher life forms, is a by-product of this reaction,” says chemist and co-author of one of the studies, Jan Kern at the Lawrence Berkeley National Laboratory in California. All animals need oxygen to breathe, including every insect, fish, and human, and most plants also require oxygen for cellular respiration.

Kern and his team extracted the protein structure that splits water, known as Photosystem II, from bacteria to study how it behaves. By bombarding these structures with lasers and x-rays, they were able to take snapshots of the process at an atomic scale, as described in the journal Nature. Another study, also in Natureused signals emitted by Photosystem II when it was hit with infrared light to study changes during photosynthesis. These detailed imaging techniques revealed that water-splitting takes place in multiple steps, which had never been observed before.

The purpose of splitting water molecules is to release electrons, which are used to power the rest of photosynthesis. “This is basically the engine that drives the entire thing,” Kern says.

With a better understanding of how these complex biochemical reactions occur, scientists not only have a more accurate picture of the engines driving life, but they could also mimic photosynthesis to generate clean hydrogen fuel.

The dream is we replace fossil fuels,” says Jenny Zhang at the University of Cambridge in England, who was not involved in the studies. “To replace it we need to source those electrons, and water is the best source.”

Eaters of water and light

Photosynthesis uses sunlight to convert carbon dioxide and water into sugar, releasing oxygen as a waste product. It evolved in single-celled bacteria over 2.5 billion years ago.

“The consequences are huge,” says biophysicist Holger Dau at the Free University of Berlin in Germany, a co-author on one of the new studies, because it “led to the oxygen-rich atmosphere we have today on Earth.” Oxygen is highly reactive and provides lots of energy to organisms that can harness it, ultimately enabling the evolution of large, active animals.

Today, photosynthesis is performed by cyanobacteria, green algae, and green plants, from grasses and wildflowers to giant redwoods. However, despite this enormous diversity of photosynthetic life, the details of the process have stayed remarkably consistent. “Nature hit on something three billion years ago and stuck with it,” says biophysicist Vittal Yachandra at the Lawrence Berkeley National Laboratory, a co-author of one of the new studies.

The first step in photosynthesis is to break apart water molecules, each of which is made of one oxygen atom and two hydrogen atoms. This is where the waste oxygen is released and the electrons are harnessed for energy.

The water molecules are split by a large enzyme—a protein structure that acts as a catalyst, speeding up biochemical reactions—called Photosystem II. Its name is a historical accident: It acts first in the process of photosynthesis, but another enzyme that comes later was discovered first.

At the center of Photosystem II is a cluster of ions, or electrically charged particles, specifically of manganese, calcium, and oxygen. This cluster is responsible for splitting the water molecules and is the focus of the new studies. “This step is insufficiently understood at the level of atoms,” Dau says.

How to break H2O

Previous studies have shown that the water-splitting reaction takes place in stages. First a water molecule enters Photosystem II and becomes bound to the metal cluster. Meanwhile, the cluster accumulates energy from incoming light, which it needs to split the water.

To find out what happens next, Yachandra, Kern, and their colleagues obtained many copies of Photosystem II from bacteria. They kept them in the dark and hit them with short flashes of laser light to drive the reactions, then bombarded them with x-rays to image how their atomic structures changed. In this way, they were able to take snapshots of the water-splitting process in action.

This revealed a surprise. “The paradigm was that the enzyme charges itself up … and then the chemistry happens in one fell swoop in the last step,” Yachandra says. But their data didn’t support this picture. “What we are finding is the last step is not one fell swoop. There are smaller steps there.”

It’s not clear what these intermediate steps are. Two water molecules may be temporarily transformed into a peroxide, in which their oxygens are joined by a new bond, forcing the hydrogens to be removed.

In the second study, Dau and his colleagues found complementary evidence. They obtained Photosystem II samples from fresh spinach leaves and stimulated them with laser flashes. Then they used infrared spectroscopy to track changes in Photosystem II, bombarding it with infrared radiation and measuring the resulting emissions to see how the enzyme was changing.

The team performed these measurements 230,000 times. “We started this experiment 15 years ago,” Dau says.

These data also suggests that there is an intermediate step in the water-splitting reaction. Dau’s team also found that the reaction was only possible because one electron and four protons moved in a coordinated way, revealing that Photosystem II exerts precise control over even the smallest particles in the reaction.

The two studies “resolved the final step of this catalysis pathway,” Zhang says. “It has been one of the biggest questions in the field.”

Evolutionary origins

It is still unclear exactly how Photosystem II, and photosynthesis as a whole, first evolved. What is clear is that the water-splitting system has stayed almost entirely the same for billions of years. “Nature has only figured out how to do it once,” Zhang says. This may be because the reaction is particularly complex, forcing molecules apart despite the bonds holding them together.

There is only one mechanism to split water molecules, and it requires a hugely elaborate enzyme like Photosystem II—so how did evolution ever hit on it?

Part of the answer is that there are other, more primitive forms of photosynthesis that don’t involve splitting water. These less-familiar versions also use light energy, but they obtain electrons from chemicals other than water, such as hydrogen sulphide, and don’t release oxygen.

Such “anoxygenic” photosynthesis is older than the more familiar kind. “You still see a lot of anoxygenic photosynthesis organisms,” Kern says, such as green sulfur bacteria, but they are confined to small niches.

Modern water-splitting photosynthesis probably evolved from these older, simpler systems. “Water is a really clever choice of a source for electrons, because water is basically everywhere on Earth,” Kern says. Using water as an energy source, “photosynthetic organisms were able to colonize virtually every habitat.”

Stealing electrons

Research into the mechanisms of natural photosynthesis could also inform the design of artificial photosynthetic systems.

Splitting water is crucial for making “green hydrogen.” The idea is to use hydrogen as a fuel, replacing some fossil fuels. But to make it truly sustainable, the hydrogen must be created by splitting water molecules. “This is a really difficult reaction,” Zhang says, so clues from nature will be vital.

A better understanding of photosynthesis could also lead to new ways of generating energy from plants themselves, taking the electrons from the cells as they perform the reaction.

In a study published in March, Zhang and her colleagues demonstrated that they could extract electrons from Photosystem II fractions of a second after it was activated by light. “We’re now beginning to see that we can wire into living systems, wire into photosynthetic organisms, so we can steal electrons,” she says.

Researchers at Imperial College London are using synthetic biology to redesign Photosystem II while keeping it embedded in living cells. Others aim to completely redesign the process using synthetic materials.

“What we are trying to do is to learn the concept of design from natural photosynthesis,” says Vachandra and Kern’s colleague Junko Yano, also at the Lawrence Berkeley National Laboratory.

Yano says the most important lesson is that the process only really works in a highly controlled environment, like that at the heart of Photosystem II. “That kind of really well-controlled chemistry, we cannot do it right now in artificial photosynthesis systems,” she says.

With more work, however, scientists may learn to mimic one of nature’s most remarkable tricks.

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