Researchers First to "See" Reactive Oxygen Species in Vital Enzyme

Using two simultaneous light-based probing techniques at the U.S. Department of Energy's (DOE) Brookhaven National Laboratory, a team of researchers has illuminated important details about a class of enzymes involved in everything from photosynthesis to the regulation of biological clocks.

The interdisciplinary team has a broad interest in flavoproteins, which were first discovered in the 1930s and derive from riboflavin, or vitamin B2. These proteins are now known to catalyze a wide range of biochemical reactions, including those that use molecular oxygen (O2) to help convert food into energy in animals, plants, fungi, and in some types of bacteria -- a process known as oxygen activation.

Although scientists have determined more than 1,200 crystal structures of flavoproteins, they've been blind to exactly what oxygen activation looks like within these enzymes. Specifically, researchers have been unable to determine the structure of the flavoprotein's reactive oxygen intermediate, a molecular complex that often forms halfway through important biochemical reactions. These intermediates possess high chemical potential energy, which is necessary to complete many critical but difficult-to-catalyze reactions in biology. Such
intermediates typically have a lifetime of only a few milliseconds and are therefore very hard to observe using traditional synchrotron methods.

"Flavoproteins represent one of only a handful of ways that nature activates molecular oxygen, a process that's important for all life on the planet," says Brookhaven biophysicist Allen Orville. "We've determined structures of some oxygen intermediates involved in several important enzymes that assist in this process. But no one has ever seen an oxygen intermediate attached to the flavin. Until now."

As reported in the Jan. 9, online edition of Biochemistry, Orville and colleagues from Georgia State University, Georgia Institute of Technology, and the University of Miami have used a new facility at Brookhaven's National Synchrotron Light Source (NSLS) to identify two possible oxygen intermediates in the flavoprotein, choline oxidase.

The researchers accomplished their work by combining two popular synchrotron techniques -- x-ray diffraction and optical absorption spectroscopy -- into one setup. By shining beams of powerful x-rays and visible light on the same region of the crystallized flavoprotein, two different but complementary sets of information are received. This allows the scientists to correlate the electronic structure of the enzyme -- which gives details about chemical activities -- with its three-dimensional atomic structure.

"The ability to collect multiple types of data from the same sample at the same time is a unique opportunity," Orville says. "It takes less time and it means you never have to move the sample and risk altering it in any way. It also removes many potential ambiguities that either technique alone cannot resolve."

To stabilize the flavoprotein intermediate, the researchers kept it at an extremely low temperature -- about -280 degrees Fahrenheit. When exposed to the x-rays, the cold flavoprotein rapidly accepts electrons liberated in the sample by the x-ray beam. This starts the enzyme reaction, which progresses a bit further and then becomes trapped in its reactive intermediate state. Using the combined data, the group identified two possible intermediate structures. Further experiments will help determine which is the true intermediate.

Orville is installing additional complementary techniques at the NSLS. Planning also is underway for several beamlines with multiple complementary techniques at the National Synchrotron Light Source II, a new, proposed Brookhaven facility that will produce x-rays up to 10,000 times brighter than those at the NSLS. The hope is to provide a means for researchers to simultaneously obtain three or four different types of data from one sample.

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Physicists Receive Patent for Improved Cancer Therapy Device

Four physicists at Brookhaven National Laboratory have been awarded a U.S. patent for the design of a "medical synchrotron" capable of delivering precision doses of proton radiation to cancerous tumors with minimal damage to surrounding healthy tissue. The new device would be more precise and less costly than existing proton-therapy systems, potentially increasing the availability and benefits of this treatment for caner patients worldwide. The Brookhaven scientists are now seeking industrial partners to license and commercialize the technology.

"In the realm of cancer treatment, proton therapy is considered 'surgery without a knife' because proton beams can deliver cell-killing energy with extreme precision, unlike conventional x-ray radiation therapy," says Brookhaven physicist Stephen Peggs, one of the lead scientists on the project. Peggs, while working at the Fermi National Accelerator Laboratory, witnessed the completion of the nation's first hospital-based proton-therapy synchrotron, installed at
California's Loma Linda University Medical Center in 1990.

"Almost as soon as the Loma Linda synchrotron went out the door, we started thinking about ways to build a better machine," Peggs says. The current design -- developed and refined as Peggs and other physicists worked on large-scale accelerators for physics experiments, including the Relativistic Heavy Ion Collider (RHIC) at Brookhaven Lab -- is the culmination of that effort.
"Our new design has improvements in beam-focusing technology to make the smallest possible beam size -- that is, the sharpest possible 'knife,'" says Peggs. Because smaller beams deliver radiation with increased precision, this improvement could have a significant impact by shortening the duration of treatment, increasing its effectiveness, or both. The new design also promises to be less costly and more reliable, which should increase its availability.

How it works

The idea behind radiation therapy is to deliver a lethal dose of radiation to cancerous cells. In conventional x-ray radiation therapy, many healthy surrounding cells are also exposed to the radiation because x-ray beams deposit their energy as they travel through tissue. In fact, most of the dose of x-rays is deposited near the surface of the body. Though cancerous cells tend to be more susceptible to the damaging effects of radiation (or less able to repair it), the collateral damage to healthy tissues limits the dose physicians can use to destroy the tumor.

Proton therapy offers an advance over conventional x-rays because proton beams deposit most of their energy where the beam stops. The original proton therapy synchrotrons were designed to deliver cell-killing doses of radiation to tumors in three dimensions by aiming proton beams from multiple directions to stop at the depth of the tumor tissue. That precision targeting allows doctors to deliver higher doses to the tumor cells while sparing healthy surrounding tissue.

But accelerators are often costly to build and difficult to maintain, explaining why the design principles for hospital-based accelerators must be radically modified, and why relatively few hospitals have them. The new accelerator design developed by the Brookhaven team offers two main advantages: "rapid cycling" and "strong focusing."

Rapid cycling allows proton beams to be injected and extracted from the synchrotron in just one turn around the circular particle accelerator. Unlike the earlier machines, which required multiple turns, this eliminates the need for sensitive feedback systems to control the beam currents, the researchers say.

"This makes the machine more robust and reliable to operate. It's more of a turn-key operation," Peggs says. "Turn it on and it consistently starts up like a transformer, rather than booting up like a PC."

Strong focusing refers to the ability to shape the proton beam and keep it focused to pinpoint dimensions. In contrast to the Loma Linda machine, where beams measure up to a centimeter across, the new design can achieve beams as narrow as one millimeter.

Pinpoint accuracy reduces collateral damage and allows physicians more flexibility in the doses they use. Higher doses could yield more effective therapy, possibly in fewer treatments.

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