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July 14, 2004
Spinach, Or The Search For The Secret Of Life As We Know It

Consider Earth four billion years ago: a high-pressure carbon dioxide atmosphere, an acidic ocean with no dissolved oxygen, a surface blasted with unfiltered ultraviolet light. Somehow, life took hold.

Some photosynthesizing bacteria found a way to survive the harsh environment of primitive Earth by secreting tough films. Accumulating mats of bacteria formed stony pillars called stromatolites, among Earth's oldest fossils but still growing today in areas like Shark Bay, Western Australia.

For a billion years the first single-celled organisms protected themselves from searing solar radiation by hiding in or under rocks or by secreting tough films. Able to fix carbon through reactions powered by UV light and catalyzed by iron and sulfur, they lived on a diet of methane, hydrogen, and bicarbonates.

By two-and-a-half billion years ago, something remarkable had happened: oxygen had begun to accumulate in the atmosphere. Bacteria similar to modern cyanobacteria had stumbled upon a way to break water into molecules of oxygen and hydrogen ions, freeing electrons and releasing energy to power their growth.

At the heart of the process was the manganese complex, a tiny molecular structure incorporating four manganese ions, one calcium ion, and a number of oxygen atoms. (Ions are atoms with net electrical charge, in this case positive charges.) Vittal Yachandra and his colleagues in Berkeley Lab's Physical Biosciences Division were the first to establish that the manganese and calcium ions are all part of the same complex, held together by bridges of oxygen atoms.

"The manganese complex produced all the oxygen that today's life forms depend on," says Yachandra, who heads the Lab's ongoing research program into the unique biological mechanism called the oxygen-evolving complex (OEC). "It changed the course of evolution and made life as we know it possible."

Gourmet spinach

The oxygen-evolving complex is housed in an assembly of proteins known as photosystem II (PSII), long the focus of attention of Yachandra and his colleagues, among them Kenneth Sauer, nominally retired yet an active member of the OEC research program.

PSII is one of several protein assemblies that function cooperatively in photosynthesis. In PSII, energy captured from light is used to split water into oxygen molecules and hydrogen ions, freeing electrons in the process. The freed electrons are transported to another protein assembly, photosystem I, then to the series of biochemical reactions known as the Calvin cycle — named for Berkeley Lab's Nobel-Prize-winning chemist Melvin Calvin — where they fix carbon and energize growth.

These engines of photosynthesis are located side by side in some bacteria and in higher plants, in special membranes called thylakoids (from the Greek word for sack). In higher plants, thylakoid membranes are contained inside organelles called chloroplasts; once these were free-swimming bacteria, but at some point in the distant past they formed symbiotic relationships with plants.

Photosystem II uses solar energy to break two molecules of water (H2O) into one oxygen molecule (O2) plus four hydrogen ions (H+), meanwhile freeing electrons to drive other reactions. At the heart of the oxygen-evolving complex is a cluster of manganese and calcium ions and bridging oxygen atoms; a possible configuration is illustrated here. (After Fufezan and Sutliff, Science)

The oxygen-evolving complex employed by primitive cyanobacteria was so efficient it is still used by plants today, including that long-time favorite of the photosynthesis researcher, spinach. "We're spinach experts, real gourmets," Yachandra remarks with a smile. "We buy 10 or 12 bunches at a time. Sometimes we shop at Andronico's, sometimes at the Berkeley Bowl, always looking for the freshest bunches."

The OEC functions even after the spinach leaves have been pulverized into juice. The sequential steps of the OEC's process can be triggered by precisely timed flashes of laser light, and after each step the solution is frozen to stabilize that specific state. In these spinach Popsicles photosynthetic structures and processes can be studied by many techniques; one of the most important, electron paramagnetic resonance, was first applied to photosynthesis in 1954 by Melvin Calvin.

An arsenal of x-ray spectrometries is also available for directly detecting manganese, calcium, and other metal atoms in PSII. X-ray absorption near-edge structure (XANES) detects how many electrons the manganese cluster's atoms have lost. Extended x-ray absorption fine structure (EXAFS) provides information about the cluster's neighboring atoms. Resonant inelastic x-ray scattering (RIXS) was developed at Berkeley Lab's Advanced Light Source by Stephen Cramer of the Physical Biosciences Division, specifically to study metal-containing proteins.

Cycling through PSII

More than two dozen proteins make up PSII, some of them very large, others small, and many of them penetrating all the way through the membrane in which the system is embedded. Deep in the heart of this nest of proteins lies the manganese cluster, whose precise arrangement of atoms remains one of biology's outstanding problems.

So complex is PSII itself that protein crystallographers were unable to image its structure until 2001, despite decades of trying. Only in March 2004 was its structure published at the relatively high resolution of 3.5 angstroms (an angstrom is a ten-billionth of a meter) — high, but still too low to see individual manganese atoms.

"It is going to be very difficult to see the manganese complex in action with x-ray crystallography," says Yachandra. Nevertheless, the details of the new crystallographic structure confirm the discovery of Yachandra and his colleagues that the complex consists of four manganese ions and one calcium ion held in close proximity by bridging oxygen atoms.

To split water, the oxygen-evolving complex cycles through a series of steps, labeled S0 through S4. The first three steps of this cycle, known as the Kok cycle, are well understood, but a persuasive model of the fourth step, S3, has only recently resulted from research by Yachandra and his colleagues. Details of the fifth crucial step, S4, are still a mystery.

"The complex works like a capacitor," says Yachandra. "It charges up step by step and then discharges in one fell swoop to create an oxygen molecule."

In the initial S0 state, two of the four manganese ions in the complex have a four-fold positive charge (MnIV), meaning they are lacking four electrons, while the other two are plus-three (MnIII) and plus-two (MnII) respectively. Moving to step S1, another electron is freed, so that now the complex has a pair of MnIVs and a pair of MnIIIs. (Electron loss is called oxidation, whether or not oxygen is involved. Electron gain is called reduction.) Moving to S2, yet another electron is freed, leaving three MnIVs and one MnIII.

Energized by light, positive charge builds as ions and atoms in the manganese cluster lose electrons in a repeating process called the Kok cycle; eventually water is split into oxygen and hydrogen. Not all steps in the process are well understood.

An electron is also freed during the transition to the S3 state. Yachandra and his colleagues propose that this electron comes not from a manganese ion but from one of the bridges among them, which contain oxygen atoms.

"The substrate-water oxidation occurring at this transition provides the trigger for the formation of the oxygen-oxygen bond, the critical step in the water oxidation reaction," says Yachandra. This is the final step in the cycle, S4, and it remains a subject of lively debate. "How the O-O bond is made is still an open question."

Having subjected spinach in solution to precisely timed flashes of laser light to advance PSII through the steps of the cycle, the researchers now reset it to zero. "If you put PSII in the dark, it goes back to the basic state," Yachandra says.

Photosynthetic timescapes

Yachandra notes that PSII "is one of nature's most complicated enzymes — complicated partly because it involves a four-electron oxidation process resulting in four intermediate states and the concurrent chemistry of O-O bond formation." He suspects that characterizing the Kok-cycle steps merely as changes in the oxidation state of manganese may be too simplistic to do justice "to the way nature tweaks the electronic states."

The prospect of more accurate modeling of geometries and electronic structures came a step closer in 2001, when the European team that first successfully determined the structure of PSII with x-ray crystallography, headed by Horst-Tobias Witt and Wolfram Saenger, provided Yachandra and his colleagues with crystals.

"We can now do x-ray spectroscopy on PSII crystals themselves," says Yachandra. "X-ray crystallography works by creating electron-density maps at various orientations" from which structural images can be derived. "We can fit our very high-resolution models of the manganese complex into these maps. Combining the two methods, we'll get the manganese complex structure at very high resolution. These are exciting times."

The rewards will be substantial. On the purely intellectual front, Sauer and Yachandra have investigated the evolutionary origin of the manganese complex. "It's fascinating that there's been no improvement in the system in over 2.5 billion years," says Sauer. "Photosynthetic bacteria used the same complex. Where did it come from?"

Clusters resembling the oxygen-evolving complex occur in tunnel-structured manganese oxide minerals, found near sites of active chemistry like underwater thermal vents. Hollandite, for example, exhibits 18 different configurations of manganese (blue) and oxygen (red) compatible with the manganese clusters of photosystem II. Primitive organisms could have used mineral clusters to aid photosynthesis.

They believe the answer may lie in the rocks. They examined manganese oxide minerals with x-ray spectroscopy and found that many contain crystalline structures corresponding to possible configurations of the PSII manganese cluster. Indeed, a single representative mineral, hollandite, exhibits a complete set of the manganese cluster's possible geometric arrangements.

Such minerals are found near deep-sea vents and on the ocean floor, and in soils, rock varnishes, and weathered outcrops. Primitive bacteria could have adapted the machinery in the rocks to help oxidize water, eventually stumbling upon a way to encode genes for manganese oxide structures in their own genomes. Just by surveying the numerous mineral structures, Sauer and Yachandra found several new candidates for the manganese cluster in PSII.

The history of the evolution of plants is fascinating in itself, but understanding the way the oxygen-evolving complex works will have practical applications as well. "You can isolate PSII from a bunch of spinach, put it in water, turn on a light, and get oxygen," says Yachandra. "We still don't know how to do that on the lab bench using synthetic catalysts."

Oxygen molecules, O2, are one product of the OEC; another is hydrogen ions (protons), H+, and electrons (e-). If a way could be found to modify the OEC to produce molecular hydrogen, H2, "we could use this in the new hydrogen-power economy," says Yachandra.

"We still don't know how to copy nature," says Sauer. "But we now seem closer to a full understanding of PSII than at any time in the last 15 years."

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