Nature's blowtorch is an enzyme system that oxidizes toxins, drug molecules, and other noxious molecules found in the body ready for excretion. But, its precise mode of action has remained a mystery. Now, US researchers have turned to solid state NMR to reveal important structural details of this system, their finding could help explain the body's response to toxins from bacterial infection or the side effects of certain drugs.

Ayyalusamy Ramamoorthy, Lucy Waskell, and Ulrich Dürr, at the University of Michigan, Ann Arbor, point out that nature's blowtorch is composed of two enzymes working together. To stretch the blowtorch metaphor, the first enzyme, cytochrome P450, is the "acetylene", which does the actual toxic breakdown work, to the "oxy" of the second enzyme, cytochrome b₅, together they are the biomolecular equivalent of the oxy-acetylene blowtorch.

To complicate matters, the two proteins can interact only when both are bound to a cell membrane, which, as any structural biologist knows, precludes an X-ray crystallographic analysis. Crucially, removing the cytochrome b^₅ from its membrane for X-ray analysis requires separation at the site active in controlling the interaction with cytochrome P450. So, crystallography can offer interesting information about the structure of the blowtorch, but it cannot reveal details about the business end.

Solid state NMR spectroscopy, in contrast, can produce detailed images of proteins in their membrane environment. But, even this powerful, emerging technique was presented with difficulties by cytochrome b₅, which itself has three components, all behaving differently. It has a rigid portion that buries itself into the cell membrane, a highly mobile, water-soluble portion, and a less mobile "linker" that connects the two.

"The challenge was something like having a room full of people and trying to get good photos of every one of them," explains Ramamoorthy, "With one picture, you probably can't do it. But if you say, 'Everyone over age 50 stand up,' and you take one picture, and then you ask for another age group and take another picture, and so on, you have a better chance." Magic angle spinning was the key to separating out the components and the team has now coined HIMSELF, heteronuclear isotropic mixing by separated local field spectroscopy. "With the techniques we designed, we were able to observe the rigid portion separately from the highly mobile and less mobile portions," Ramamoorthy adds.

The team published details of the membrane-spanning segment of cytochrome b₅ in the Journal of the American Chemical Society. They revealed for the first time the helical structure and how this component tilts relative to the membrane. In the latest work, published in BBA Biomembranes, the team has demonstrated that once both molecules are bound to the membrane, cytochrome b₅ modulates the overall motion and the structure of cytochrome P450.

"Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅," the researchers say. The same NMR techniques should be equally applicable to studies of other membrane-bound proteins.

Related links:

• BBA Biomembranes, 2007, in press
• J Am Chem Soc, 2007, 129, 6670-6671
• Ramamoorthy Page

Article by David Bradley

P450 and b5 coupling revealed by HIMSELF in a membrane