Single molecule Studies of Flavoproteins


Oxygen is a powerful and versatile oxidizing agent that can be used to create a wide variety of chemical structures.  However, oxygen’s very versatility is also a danger to the cell as oxidation reactions are generally irreversible and side reactions can generate toxic byproducts, such as hydrogen peroxide and free radicals. Free radicals in turn have been implicated in a wide variety of aging related disorders. In order to avoid the improper oxidation of substrates oxygenases must have control mechanisms to ensure that oxygen reacts only under controlled conditions.

The family of flavoprotein monoxygenases represented by p-hydroxybenzoate hydroxylase (PHBH) offer insight into how such controlled is obtained. PHBH is a homodimer with two identical subunits each non-covalently binding one molecule of FAD in the active site. The environment of the enzyme surrounding the flavin in PHBH is crucial to the efficiency and regulation of the reaction. The flavin C4 (a)-hydroperoxide, the key intermediate in the catalytic cycle, is unstable in protic solvents and must be shielded from solvent during this phase of the reaction to avoid the formation of toxic hydrogen peroxide. However, the active site must be exposed to solvent in order for the substrate to bind. A different environment around the active site is therefore required for each step of the reaction. In addition, PHBH must distinguish between similar substrates as 4-aminobenzoate, a close chemical analogue of p-hydroxybenzoate, is an important intermediate in folic acid synthesis and must not be reduced by the enzyme.

Crystallographic studies on PHBH have suggested that PHBH achieves this control over the reaction environment by the flavin adopting different conformations in each step of the reaction. The crystal structure of PHBH shows the isoalloxazine ring of the flavin in three conformations depending on the crystallization conditions. When the wild type protein is crystallized with its natural substrate, p-hydroxybenzoate, the isoalloxazine ring is buried in the inside of the protein and is shielded from solvent (the “in” conformation of the flavin). Under other conditions the flavin crystallizes in what is called the “out” conformation where the isoalloxazine ring of the flavin is exposed to solvent and tilted away from the substrate binding site towards the surface of the protein.

Figure 1.

Kinetics of Transition =>
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