Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to their corresponding aldehydes. They are members of a select group of enzymes that contain cofactors derived from the post-translational modification of specific active site residues. In CAOs, that cofactor is 2,4,5-trihydroxyphenylalanine quinone (TPQ), derived from a precursor active site tyrosine (Y405 in the CAO from H. polymorpha, HPAO). Replacing the precursor tyrosine with unnatural amino acids provides a subtle method to perturb and probe the biogenesis pathway. Para-aminophenylalanine (pAF) was incorporated into position 405 of HPAO using the in vivo stop codon suppression method developed by Peter Schultz (Scripps). By incorporating pAF, we can test the importance of pK_a vs. redox potential for the formation of a charge transfer complex that is essential for TPQ biogenesis. The purified protein (Y405pAF) runs as a single band on SDS-PAGE and is metal-free. Incorporation of pAF at position 405 was confirmed by mass spectrometry. When reconstituted aerobically with Cu(II) at pH 9, a peak grows in with a λ_max ~450 nm (WT HPAO λ_maxTPQ = 480 nm). This new species does not react with phenylhydrazine to form a hydrazone, suggesting that it is not a quinone. In addition, reconstituted protein shows no activity towards benzylamine or ethylamine. When the protein is reconstituted with Cu(II) anaerobically, a similar 450-nm peak appears. No further spectral changes are observed after subsequent exposure to air, suggesting that the addition of Cu(II) alone is sufficient for formation of the 450 nm-species.

Further spectroscopic characterization was carried out on Y405pAF using resonance Raman, EPR, and X-ray absorption spectroscopies in order to detect an organic-based, aniline radical or formation of Cu(I) due to electron transfer from aniline to Cu(II). While these results were not definitive in identifying the 450-nm species, x-ray crystallography of Y405pAF revealed that the aniline ring was unmodified upon addition of Cu(II) and that the aniline was liganded to the metal center, in position to undergo inner sphere electron transfer or to form a charge transfer complex. In addition, single crystal UV-Vis spectrophotometry demonstrated that the reconstituted crystals had similar absorbance at 450-nm as was seen in the solution reaction.

pAF has also been incorporated into position 305, a strictly conserved active site tyrosine in WT HPAO that controls runaway oxidation during cofactor biogenesis and ensures proper cofactor formation. Both aerobic and anaerobic reconstitution of the Y305 variant with Cu(II) produce the same peak at λ_max~450 nm, analogous to what was seen with Y405pAF. While the O-4 of Tyr405 is only ~2.5 Å from the metal center in the precursor structure, the O-4 of Y305 is ~4.9 Å away, too far to undergo inner sphere electron transfer with the metal. Thus, the phenomenon we observe with the Y305pAF protein might be instead outer sphere electron transfer from the Cu center to the aniline group, possibly mediated by active site water molecules.

In order to study the effects of second sphere ligands, pAF was incorporated into position 407, a conserved tyrosine that hydrogen bonds with one of the histidine ligands to the Cu. Unlike Y405pAF and Y305pAF, Y407pAF does form TPQ upon reconstitution with Cu(II) and does turn over benzylamine. Based on a preliminary kinetic characterization, Y407pAF is less reactive toward benzylamine than holo-WT-HPAO. These studies pave the way for a more complete kinetic characterization of Y407pAF and demonstrate how unnatural amino acids can be used to tune active site metals as second sphere ligands.

Overall, our work on incorporating unnatural amino acids into HPAO has been very provocative, both for elucidating the mechanism of the CAOs and for introducing a new tool to prove the behavior of the active site metal in copper-dependent enzyme reactions. Future work will continue to address how the CAO active site catalyzes the formation of a single post-translationally modified product and test the plasticity of this active site in tolerating deviations from the natural configuration.