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A Crystal With A Story to Tell

Biochemistry: Single crystal traps elusive intermediates in O2 activation by a nonheme iron enzyme

by Celia Henry Arnaud
April 23, 2007 | A version of this story appeared in Volume 85, Issue 17

Credit: Courtesy of John Lipscomb
Credit: Courtesy of John Lipscomb

A new crystal structure answers questions about the mechanism of a nonheme iron enzyme by providing simultaneous snapshots of several oxygen-containing intermediates.

Iron-containing enzymes are major players in activating molecular oxygen for reactions with organic substrates. One of the questions concerning iron enzymes that lack porphyrin-containing heme groups is: How does O2 interact with the iron and then attack substrates?

Biochemistry professor John D. Lipscomb and postdoc Elena G. Kovaleva of the University of Minnesota have answered this question for one particular nonheme enzyme. In a single crystal of a dioxygenase that cleaves 1,2-dihydroxyaromatic compounds such as catechol, they captured the structures of three intermediates???two of which had not been seen before???that are involved in O2-activation and -insertion reactions catalyzed by the enzyme (Science 2007, 316, 453).

On the basis of spectroscopic studies, Lipscomb previously predicted that the enzyme's iron atom binds O2 and the catechol substrate simultaneously, with the resulting complex serving as a conduit for electron transfer from the organic substrate to the dioxygen. But observations of the key intermediates specified by the proposed mechanism proved elusive.

In the first newly observed intermediate structure, the oxygen molecule binds to the iron in a side-on fashion (rather than the expected end-on configuration) to give a superoxide (O2• –) ligand, while the two oxygens of the catechol substrate bind to two adjacent ligand sites on the iron.

The key feature of this intermediate is the buckling of the aromatic ring. With one of the catechol's hydroxyl groups pushed out of the plane of the ring, "the aromaticity has been partially compromised in the system," Lipscomb explains. "This is telling us that one electron has already left the catechol ring and gone through the iron to the dioxygen."

Next, the iron-bound superoxide attacks the catechol moiety to form an alkylperoxo intermediate. "We actually see the direct connection of the iron through both oxygens of the O2" directly to a ring carbon of the substrate, Lipscomb says.

A stroke of luck allowed Lipscomb and Kovaleva to trap the intermediates in a single crystal. Kovaleva found a new way to crystallize the enzyme so that the four nominally identical active sites experience different packing forces that create slight differences among them. "We can't actually see the differences in the structure of the active site," Lipscomb says, "but we see that the catalytic cycle has effectively stopped at different points in each subunit."

The work is a "tour de force," says Julie A. Kovacs, a chemistry professor at the University of Washington, Seattle. "The fact that they have been able to observe all of these intermediates in a single crystal is truly amazing."

In a related paper describing another nonheme enzyme, Dominique Bourgeois and coworkers at the Institute for Structural Biology in Grenoble, France, combine data from X-ray crystallography and Raman spectroscopy to suggest that peroxo intermediates bind end-on in superoxide reductase, which is involved in the oxidative stress response in anaerobic organisms (Science 2007, 316, 449). Michael K. Johnson, codirector of the Center for Metalloenzyme Studies at the University of Georgia, comments that this work "represents a major advance in understanding the mechanism of biological superoxide detoxification." But he points out that the proposed intermediate contradicts recent spectroscopic studies of the same peroxo adduct that were interpreted in terms of a side-on species.


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