PUTTING THE OH WHERE IT BELONGS | June 14, 2004 Issue - Vol. 82 Issue 24 | Chemical & Engineering News
Volume 82 Issue 24 | p. 14 | News of The Week
Issue Date: June 14, 2004

PUTTING THE OH WHERE IT BELONGS

Study shows how P450s hydroxylate hydrocarbons but not themselves
Department: Science & Technology
Green
Credit: PHOTO BY SUELLEN HOPFER
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Green
Credit: PHOTO BY SUELLEN HOPFER
IRON WILL
The cysteine that binds the heme iron in P450s allows these enzymes to oxidize inert substrates.
Credit: COURTESY OF MIKE GREEN
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IRON WILL
The cysteine that binds the heme iron in P450s allows these enzymes to oxidize inert substrates.
Credit: COURTESY OF MIKE GREEN

Only a handful of heme proteins—for example, the medically important family of cytochrome P450 enzymes that metabolize drugs in the body—are capable of attaching hydroxyl groups to inert hydrocarbon substrates. A new X-ray absorption study of a model member of this protein class hints at what allows these enzymes to create reactive iron intermediates that can perform this demanding chemistry without destroying themselves.

The active sites of cytochrome P450s contain a heme cofactor whose iron center is coordinated to the protein via a cysteine thiolate. During catalysis, these enzymes are thought to use dioxygen to generate an Fe(IV)=O radical species. This highly reactive ferryl radical species is thought to abstract hydrogen from the hydrocarbon substrate, forming a protonated ferryl that then hydroxylates the substrate.

Somehow, this highly reactive ferryl radical species performs hydroxylations without oxidizing the surrounding enzyme. “That’s a remarkable feat,” says Michael T. Green, an assistant professor of chemistry at Pennsylvania State University. He collaborated with chemists Harry B. Gray of California Institute of Technology and John H. Dawson of the University of South Carolina to try to figure out what allows a P450 to hydroxylate hydrocarbons without destroying itself.

Since it has proven impossible to trap and study these ferryl radical species in P450 enzymes, Green’s team turned to chloroperoxidase—a closely related fungal enzyme with an identical active site—as a model system. Using X-ray absorption spectroscopy to estimate bond lengths, they show that chloroperoxidase uses a catalytic intermediate just like the protonated high-valent Fe=O species thought to participate in P450 catalysis [Science, 304, 1653 (2004)].

This unexpected observation “gives us an indication of how P450 enzymes manage to hydroxylate their substrates,” Green says. He suggests that the way the heme iron is tethered to the P450 enzyme is key: Most heme enzymes use a histidine to coordinate iron. But P450s’ cysteine thiolate linkage donates electron density to the ferryl group, making the oxo moiety more basic and ensuring that the species is protonated at pH values lower than 8.2, his team finds.

“This is a key contribution to the ongoing debate about the mechanism of P450,” says chemistry professor James M. Mayer of the University of Washington, Seattle. His early studies of synthetic model systems indicate that the reactivity of ferryl species depends on both their redox potential and the ease with which their reduced forms can be protonated.

So because the electron-donating thiolate linkage makes it easier to protonate the ferryl species, P450s can rely on ferryl radicals whose redox potentials are lower than what would normally be required to hydroxylate inert hydrocarbon substrates, Green says. “Such species, while capable of hydroxylating normally inert saturated hydrocarbons, are less likely to oxidize the enzyme itself,” he adds.

 
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