Laser/X-ray experiment plumbs secrets of nitric oxide

Researchers at the University of Buffalo (UB; Buffalo, NY) are using a novel technique called photocrystallography to investigate how nitric oxide (NO) bonds to metal atoms. In blood, NO is produced by the enzyme nitric oxide synthase and binds to hemoglobin's iron atom. The molecule performs crucial roles in blood chemistry, including constricting and expanding blood vessels. Vasodilation, in particular, is initiated by NO activation of the enzyme guanylate cyclase. (Viagra works by controlling the action initiated by NO.)

The research has overturned the assumption, widely held for the past quarter-century, that when compounds formed by NO bonding to a metal atom are illuminated with laser light they are only electronically excited. In fact, "small molecules like NO combine to transition metals in novel ways," says Professor Philip Coppens at UB. His group used a technique to show that the illuminated molecules shift, briefly, into a different atomic arrangement, thus becoming different molecules.

The NO-metal combination forms linkage isomers. Nitrosyl compounds (in which NO is bound to a transition metal) can be formed either by NO binding sideways to two iron atoms, or the NO can invert and combine through oxygen to form isonitrosyl.

The researchers want to understand the kinetics of NO uptake and release (which occurs before vasodilation) and the different binding modes of NO. Coppens declined to discuss how this information might be useful for clinical applications. Coppens is working with George B. Richter-Addo at the University of Oklahoma (Norman, OK) and Kimberly Bagley at Buffalo State College (NY) on biological applications.


Because a crystal has a well-defined and periodic atomic arrangement, it acts as a complex grating that diffracts the x-rays. By recording and analyzing the diffraction pattern, researchers can extract information about the molecular structure of the crystal.

Photocrystallography provides information about the molecular structure of the crystal after it has been excited by a UV laser pulse. Coppens' group uses a tiny crystal of the ground state material and holds it at cryogenic temperatures. They excite the molecules in the crystal with light from different types of laser, depending on the specific sample being studied. For some experiments, they have used a tripled Nd:YAG laser (at 355 nm). A high-intensity x-ray pulse from a synchrotron then passes through the crystal. The diffraction pattern caused by this quick burst of x-rays provides information about the bonding patterns in the crystal at that moment.

Because nitrosyl compounds have relatively long lifetimes at cryogenic temperatures, they can be illuminated for several hours so that a large fraction of the molecules can be converted to the photo-excited state.

Stroboscopic Experiments

For the study of much shorter-lived transient species, the x-ray beam is turned into pulses using a brass wheel with slits in it. By controlling the rotation (and slit design) of the wheel, the researchers can control the duration and frequency of x-ray pulses. The process is repeated thousands of times per second in a stroboscopic experiment.

The number of photons needs to be comparable to the number of molecules in the crystal to excite most of the molecules without heating the crystal until it evaporates. The laser produces intense UV pulses of about 200 mJ/pulse, which are then fed into a fiber. A tapered fiber delivers the laser light to the tiny crystals, about 50 mm on a side. The pulse loses about half its energy in the fiber. The bundle of fibers around pump fiber collects fluorescent light from the crystal. This provides diagnostic information about the lifetime of the excited state and the health of the crystal.

The researchers are working with synchrotrons at Brookhaven and plan to work with the Advanced Photon Source at Argonne National Laboratory. The x-rays they work with have a wavelength of 0.64 �, corresponding to energy of roughly 20 kV. Chris Kim, Lynn Ribaud, and Guang Wu at UB have developed the system at Brookhaven. Kim and Sebastian Pillet worked on analyzing the diffraction pattern.

The stroboscopic method excites the crystal and then gates the x-ray probe to different times, which allows researchers to watch the evolution of these short-lived species. Eventually, they may be able to use the time structure of the synchrotron for the stroboscopic technique.

Yvonne Carts-Powell, contributing editor, Medical Laser Report

Source: Medical Laser Report, Pennwell Inc., June 2001

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