Biomimicry

by Janine M Benyus

  Still, when Woodbury looks at these bacteria, the picture in his mind’s eye is much more detailed than what the rest of the world sees from the new molecular maps. Even before the maps were drawn, he and other geneticists had been probing purple bacteria with their own set of tools, sequencing the proteins and making deductions based on carefully controlled mutations. “I know every amino acid in that protein pocket,” says Woodbury. “But knowing what they are and knowing what they do are two different things. These days, we want to go beyond mere structure. We want to know how structure affects function—what exactly makes it work so well. I find this out by distorting or even ‘turning off’ one piece of the structure at a time, through a process called mutagenesis.” Simply put, Woodbury uses biotechnology to create mutant bacteria with a specific defect in their reaction centers. “The question we ask is, how does this specific change affect their ability to photosynthesize? That’s how we learn which parts of the reaction center are most important.”

  The purple bacterium cooperates in a notable way, which is what makes it such a model organism to work with. Not only is it a simple system, consisting of just one kind of reaction center, but it is also ambidextrous when it comes to garnishing energy from its world. It can photosynthesize one moment, then switch to oxidizing its food through respiration just like we do. As Woodbury says, “That flexibility means we can tinker with its photosynthetic mechanism, and even impair it a bit, without running the risk of killing the patient.”

  I try to imagine the size of its reaction center, given the fact that it is tucked into a membrane of a bacterium that Woodbury tells me is only one to three microns long. Several thousand of these bacteria could fit into the period at the end of this sentence. Now imagine that the reaction center inside this bacterium is itself only about thirty angstroms by eighty angstroms. An angstrom is one tenth of a billionth of a meter. If you had a string of beads, each one an angstrom, and you wanted to span an inch, you would need to string together 250 million beads. Now tick off the first thirty of those beads, and you have an idea how wide the reaction center is. Tick off eighty, and you’ve got the reaction center end to end.

  “An electron moves down one side of that wishbone-shaped reaction center at a speed that is equally astonishing,” says Woodbury. “It’s measured in picoseconds—trillionths of a second.” To comprehend this tiny number, consider that one picosecond is 1 × 10-12 seconds and the age of the Earth is about 1 × 1012 days. That means a picosecond is to a second as a day is to the age of the Earth. And it takes only a few hundred of these picoseconds for an electron to make it from the inside of the membrane to the outside. By the time you can form a thought, charge separation could have happened many millions of times. How do you spy on a molecular complex that small, and capture a process that fast in the act?

  The answer to the size question is that you don’t spy on one molecular complex; you spy on a whole test tube of reaction centers at once. The trick is to give them a “start” pulse of light so they all begin photosynthesizing at the same time. That way, what’s happening at any one moment to the group is also what’s occurring in each reaction center.

  You tackle the timing quandary with ultrafast laser pulses that flash on to take the reaction center’s “picture” at various stages of electron transfer. To see this ultrafast photography for myself, I went down to the laser room where Woodbury had laid out a track for the laser light to go around. Imagine a Christmas train set with light moving around it instead of trains. There were beam splitters, mirrors, and laser beams of various colors focused on vials of purified photosynthetic reaction centers. The starting gun is a burst of coherent light (oscillating up and down in perfect step, at the same phase and wavelength). It excites the reaction centers and causes them to begin their handoff of electrons. While that happens, Woodbury probes the vial with a second beam to see what’s up.

  “At rest, every molecule will absorb at a precise wavelength and then fluoresce—emit light as it releases the energy. But when that molecule is excited by sunlight, for instance, it will change its shape and absorb and fluoresce at a different wavelength. [This is the idea behind mood rings—when the chemicals heat up and change shape, they absorb (and reflect) a different color of light.] This ‘spectral signature’ changes continuously as a molecule moves and participates in photosynthesis, the electrons hopping from one spot to the next. By tracking these changes in spectral signature, we can spy on what the molecule is doing.”

  After pulsing the vial of reaction centers with a “start” light, Woodbury dials a new wavelength on the laser and begins probing with that beam. He snaps picophotos at discrete intervals of time, looking for fluorescence. “The molecule changes shape as it moves through the reaction. We’re watching carefully, and when the molecule absorbs the probe beam and emits light, we note the time. This tells us that at one point three picoseconds, it had a spectrum like this, and was in this particular stage of the reaction. We then repeat this probing with different wavelengths of light to get a complete picture, really more of a movie, of how the molecule changes through time. The reaction centers from our mutated bacteria will function differently from wild bacteria. By comparing the movie of the mutant’s changes with the movie of a wild reaction center, we try to guess how the mutation has affected photosynthesis.”

  Woodbury induces the changes in the wild reaction center by rewriting the genetic blueprint (editing the DNA sequence). “When I pull out a piece that makes photosynthesis shut down entirely, I figure, here’s something important, and I go tell Devens and Tom.” Devens Gust puts it this way. “It’s as if Neal is digging inside a computer and removing random parts of software programs. Say we want to know what makes word processing tick. One day he removes the fonts, and we can’t type anymore. So we say, fonts must be important. Let’s go model them.”

  No one wakes up one morning and decides to model something as big as a reaction center; the quest grows organically from much humbler beginnings. Years ago, Tom and Ana Moore were hot on the trail of antenna function—the satellite dish that expands the plant’s reach. Tom had done his graduate work on carotenoids (the pigments in antennas), which at that time had not been all that well characterized or mapped. He and Ana were trying to isolate carotenoids from living systems to see how they worked, but it was proving difficult. Devens Gust, in the meantime, was working with molecules called porphyrins, which are cousins of chlorophyll and also appear in antennas.

  At lunch one day, Gust and Tom Moore, who had never worked together, began talking about their separate but mutual antenna problems. They thought, why don’t we try to hook a carotene to a porphyrin and build a simplified antenna? At that time, there were no pictures of how these components were oriented in relation to one another in real life, so Moore and Gust made educated guesses and tried to chemically bond these molecules to test their hypothesis. “It was a grueling task, and grad student after grad student gave up in frustration. Finally we hired a Ph.D. student from the University of Montana, Gary Dirks, who locked on this thing like a pit bull. He lived in the lab until he finally found a way to put them together at just the right orientation, and they worked!” They had guessed that the carotene and porphyrin would have to have their orbitals overlapping somewhat to help the energy resonate from one to the other, antenna style. Although their idea made sense to them, it went against conventional wisdom at the time. “When the pictures of purple bacteria came out, we were thrilled to see that the antenna elements in our artificial device were indeed oriented at almost exactly the same angle and distance as the real antennas,” says Moore with pleasure. “We were spot on.”

  The Dyads

  But energy transfer wasn’t electron transfer; having grabbed one brass ring made them want to try for the other. Already, other boats had pulled ahead in that leg of the race. Paul Laoch at Northwestern and a group in Japan had managed to build a dyad (a two-part molecule) that would transfer an electron from an excited porphyrin to an acceptor
called quinone. Instead of relaxing back to its old orbital around porphyrin, the excited electron suddenly had a “competing pathway”—a better offer—in the form of the orbital around quinone. This second orbital was especially inviting because it was close at hand and slightly lower in energy, like a basin in the energy landscape. The trick was to bond a donor and acceptor so that their electron orbitals overlapped.

  “It’s as if the dyad builders had dug a riverbed between the two molecules.” Unfortunately, the “slant” of the riverbed was not great enough, and it allowed the electron to flow both ways. After a brief separation of plus and minus, the electron soon found its way back and the charges recombined in a burst of heat, wasting the energy before it could be used. “They had a pretty good yield [the percentage of photons that successfully triggered charge separation], but the charge-separated state lived only ephemerally—one to ten picoseconds.” Since that’s too short to get chemical work done, it wasn’t a good mimic of photosynthesis.

  “Our task was to get the charges to separate quickly and then hold them like that—to slow down recombination. Putting some physical distance between the plus and minus charges seemed like a good delay tactic. We asked ourselves, what if we were to add another molecule to the donor-acceptor dyad and make it a donor-donor-acceptor string, a triad?” Gust and Moore had already had luck hooking carotene and porphyrin together in a donor-donor pair. By adding a quinone as an acceptor, they would create a triad. In 1979, they set their sails.

  The Triad

  On paper it looked like a straight shot to the buoy. But in the lab, winds are fickle, and nothing is as smooth as you might imagine, especially when the waters are uncharted. Dr. Ana Moore would be the actual builder of the molecule, the wet-lab chief who would put it together one agonizing organic reaction at a time.

  If Gust has the eyes of a perched hawk, then Ana Moore’s are a raven’s—cocked, curious, and piercing. Like Tom, she is riveted by her work and tells me that she dreams solutions to knotty problems in her sleep, or they occur to her unbidden in the shower. For our talk, we go out to one of the terra-cotta benches that I spied on the way in. Here we are bathed in unadulterated Arizona sunlight, contemplating a process that might make the power of that sunlight as available to us as it is to the nearby vines. Ana Moore illustrates the future as she sees it, filling my notebook with chemical graffiti.

  More than anyone else, Moore speaks with an engineer’s sensibility, as if the chemical groups, which were so hard for me to visualize, are actual bone and brick, strung together with hinges and joints. She talks me through the synthesis process in a strong Argentinean accent, speaking faster and faster as her excitement builds, like a roller coaster after the crest.

  “We decided to bind the groups together with amide bonds, which is what amino acids are joined with. Amide bonds are stable and versatile—we figured these bonds would keep our molecule strung out in a line, rigid enough so that it would not fold back on itself and mechanically recombine the charges. The only problem is that forming these kinds of bridges takes many, many steps.”

  Organic synthesis is an art, she says, like gourmet cooking. Acquiring a “good touch,” a feel for how and when to time and sequence your reactions, is not something that can be taught. You must simply do it for years and years. A good synthetic organic chemist is grown, and a team like the ASU group would be stuck in the irons of theory if it were not for a good synthesist.

  Mention Moore’s name to others in the ASU team, and you’ll hear the words wizard, magician, and miracle worker. I repeat this to her and she laughs, not understanding the fuss. “You know why I do this?” she asks me. “I love to build molecules. Once I know a compound exists in nature, I have to build it just to see if I can. But to build this one that does something, that’s even better.”

  Building a molecule synthetically means keeping track of many different reactions happening in beakers throughout the lab over many months. To prepare a chemical group for bonding, you must first give it a chemical handle that other chemical groups can home in on and bond with. While that’s happening, you’re also adding a handle to the next group you want to include. To make sure you get only the reaction you want, you must protect certain sites on the chemical groups, in a kind of masking procedure. Once everything is masked and equipped with handles, you forge your first bond. If that’s successful, you go in and deprotect the masked sites and begin all over again—adding a handle, protecting, bonding, and deprotecting. Each of these steps requires a special reactant—a chemical bath bubbling at just the right temperature for just the right amount of time. It may involve dozens of stages, adding layer upon layer until you have built your molecule.

  “If one step bombs,” she says, “you have to clean out your beakers and start all over again.

  “Right in the middle of building the triad Tom went to Paris for a sabbatical, and I got a position with the French Museum of Natural History. Devens was also working over there, along with a colleague of ours named Paul Mathis. They were all working at Saclay, a nuclear facility that happened to be the site of the largest photosynthesis research lab in Paris.” The nuclear connection to photosynthesis is not as incongruous as it sounds. Most of what is known about photosynthesis was learned by putting radioactive tracers on CO2 and then following the carbon through to its products in the leaf.

  Moore took her plans for the molecule to Paris and began building it in a bare-bones wet lab at the museum. “Every night I would bring the work-in-progress home with me and put it in the fridge. I had two small children in day care, and by the time I had gotten to the office, I had been on five subways, all with my precious vial. It took me a year and a half to finally assemble a molecule that we thought would work. The museum didn’t have the spectroscopes we needed for testing so I had to send the vial out to Saclay for Tom and Devens to test.”

  Tom Moore picks up the story. “We knew that when we irradiated it, if it was working properly, the negative charge would run one way, leaving a positive charge at the other end. The positive charge on one end would cause the assembly to absorb light at a particular wavelength, so that when we had that final product (a charge-separated state) we’d see a large jump on our detector instruments. Sure enough, when we probed it with a certain wavelength, we saw an enormously large signal. We were jumping up and down, and about to call Ana, when the technician came out red-faced and told us he had set the probe at the wrong wavelength so that what we had just seen was not accurate. It was a tremendous letdown and we thought, that’s it. It’s not going to work. But then we set the thing up correctly and, lo and behold, it worked. The signal was even stronger! Ana took the train out to Saclay, and, being the Doubting Thomas that she is, she wanted to run the control again.”

  “When I saw it with my own eyes,” says Ana Moore, “I knew it was true.”

  Perhaps the most amazing thing was how long they were able to keep the plus and minus separated in the triad. “Before this, the longest charge separation (using a dyad) had lived ten to one hundred picoseconds before collapsing together in a burst of heat. There was no way to grab hold of the potential. But with the triad, we watched the clock tick off the digits and we couldn’t believe our timing. It lasted and lasted—two hundred to three hundred nanoseconds, ten thousand to one hundred thousand times longer than the dyad! For the first time, we had sufficient distance and staying power to envision doing some real chemistry at the ends.”

  Standing in Saclay, France, thousands of miles from home, the photosynthesis mimics stared at the instruments, then at one another, then back at the instruments. They’d done it! They’d glided round the buoy in the race the whole world of photochemistry was watching. Gust presented the paper at a Gordon Research Conference in 1983, setting the pace for other boats of researchers for the next decade.

  The Pentad

  After that, triads of all shapes and descriptions were developed. Everyone was trying to better Gust’s and the Moores’ separation time and yi
eld. For their part, the trio was already thinking of ways to go beyond the triad and separate the charges even farther. Ana Moore relates, “When we came back home, we hit the ground running. We built it to a four-part molecule, and finally, we went to the pentad, a five-part donor-donor-donor-acceptor-acceptor molecule. That’s our best effort yet. With the pentad, we achieve a quantum yield of eighty-three percent, meaning that for every one hundred photons we pump into the system, eighty-three of them cause a charge separation. Photosynthesis runs at ninety-five percent, so we are creeping closer. Best of all, the charge-separated state of the pentad lasts even longer than the triad. We’re tweaking to improve it all the time.”

  The pentad’s chemical signature is written like this: C-Pzn-P-Q-Q. On the far left is a carotene, then a porphyrin molecule with zinc, then plain porphyrin, then a naphthoquinone and a benzoquinone. The donor-acceptor lineup looks like this: D-D-D-A-A. Each of these molecules has a unique shape and electronic “personality” and, therefore, a unique affinity for accepting or giving away electrons. To make sure the slant is in the right direction, so the electron doesn’t find its way back too soon, the left-to-right lineup features better and better acceptors, each with a lower profile in the energy landscape. The carotene at the far left is the best donor; it’s highest in the energy landscape and most eager to give away its electron. The quinone on the far right is the low spot in the energy landscape and therefore the best acceptor. The electron goes from one to the other like a ball bouncing downstairs, ultimately coming to rest on the last quinone.

  In the pentad, the artificial photosynthesizers have added another dimension besides pure electron transfer. There’s a tiny mimic of the leaf’s antenna in the Pzn-P pair. When light strikes the Pzn, energy, rather than an electron, heads over to the P. P then reacts to this energy by donating an excited electron to the first quinone, which in turn transfers it to the second quinone. Each positive charge or “hole” that is left is neutralized or “filled” by an electron from the molecule to the left.