Elusive Enzyme Species Trapped

Working independently and after years of unsuccessful efforts, two groups have captured and characterized critical intermediates in oxidation reactions catalyzed by two key enzyme families: cytochrome P450 monooxygenases and DNA-repair dioxygenases.

The P450 monooxygenase study helps confirm researchers’ understanding of the workings of a ubiquitous group of enzymes that play a critical role in nutrient and drug metabolism. And the DNA-repair dioxygenase report helps nail down the detailed molecular mechanism of oxidative demethylation, a process that plays a major role in epigenetic gene programming and in conditions such as obesity and cancer.

Cytochrome P450 enzymes incorporate a heme iron center and use O₂ to hydroxylate aliphatic carbon-hydrogen bonds, with the remaining oxygen reduced to water. Researchers have tried for decades to pin down a key intermediate in P450 catalysis, a postulated iron(IV)-oxo-porphyrin cation radical known as compound I.

Jonathan Rittle and Michael T. Green of Pennsylvania State University finally trapped compound I by using a P450 enzyme from a species of thermophilic archaea, a peroxide substrate, and rapid freeze-quench techniques (Science, DOI: 10.1126/science.1193478). Their electronic and kinetic characterization of the species confirms that it is, in fact, an iron(IV)-oxo compound and can perform the hydroxylation reaction.

“This is an excellent piece of work that clarifies the nature of a critical catalytic intermediate,” says Paul R. Ortiz de Montellano of the University of California, San Francisco. “Most of the earlier work involved indirect measurements and were useful but imprecise. Given the importance of cytochrome P450 in biology, medicine, and biotechnology, an understanding of the basic physical and chemical properties of its catalytic species is a major advance.”

Intermediates of DNA-repair dioxygenases also could not be observed before, because they are unstable in water after release from an enzyme. To capture AlkB catalytic intermediates, Chuan He of the University of Chicago; Qiang Cui of the University of Wisconsin, Madison; and coworkers cross-linked each of three modified-DNA substrates of AlkB to its active site. They crystallized the complexes with cofactors iron and α-ketoglutarate but without oxygen. Adding oxygen to the crystals caused oxidative demethylation of each substrate to begin but to stop early, enabling crystallographic characterization of the intermediates (Nature, DOI: 10.1038/nature09497).

Gregory Verdine of Harvard University first used disulfide cross-linking to capture conformational intermediates in DNA repair but notes that it has not been used before “to poise a complex in a precatalytic state and then observe reaction intermediates.” The intermediates He and coworkers found had been hypothesized earlier, “but other mechanisms and structures were possible,” Verdine says, adding that the study “provides a detailed glimpse into the action of one dioxygenase and by extension the class at large.”

This is “the next big step in understanding DNA and RNA oxidative demethylation,” says John D. Lipscomb of the University of Minnesota, who has used enzyme crystals to study oxygen binding. “It removes a great deal of speculation about the intermediates and establishes a likely prototype for all of the enzymes from this large and growing family that modify or remove groups from DNA, RNA, and histones. Finally, this is an excellent demonstration of the remarkable ability of enzyme crystals to slow or stop reactions mid-cycle so actual intermediates can be obtained.