An example of the catechol-cleaving type of extradiol dioxygenase is the biphenyl-cleaving enzyme from P. cepacia.

Extradiol Dioxygenase from P. cepacia (1han)	The Catalytic Site

• This enzyme functions as a monomer

• Surprise. The iron is liganded by two His, a Glu, and a water

• The water is displaced when the substrate binds

• The trick to observing substrate bound without reaction is to oxidize the iron to Fe⁺³

Catalytic Site with 2,3-Dihydroxybiphenyl Bound (1kmy)

Although these two enzymes have very little homology, their catalytic sites align remarkably well:

Backbone Alignment, 1han vs 1bou	The Catalytic Site
rmsd = 17.8	rmsd = 0.11

The benzoate oxidase is a representative of Class II enzymes, whereas the biphenyl is a Class I. The classifications are based on the extent of sequence identity.

The mechanism of action is not as well established as that for the introdiol enzymes, although the extradiols are the more common type of enzyme. Here is a reasonable guess:

Mechanism of Extradiol Dioxygenases

• A major difference from the intradiol enzymes is the binding of O₂ directly to the iron

• The coordination of the O₂ to the substrate to produce a peroxide is similar

• Note that two waters are shown coordinated to the iron in the resting state, although X-ray structures show only one clearly

Very recently [Kovaleva and Lipscomb, Science, 2007, 315 453], the intermediates labelled A and B above have been captured by using an unreactive substrate, 4-nitrocatechol, and flash freezing the crystal.

Intermediate A, O2 Sideways Coordinated	Intermediate B, Alkylperoxo Bridged