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Fig. 4. Catalytic intermediates in the oxidative half-reaction of E. coli amine oxidase. X-ray single-crystal structures [C. M. Wilmot, J. Hajdu, M. J. McPherson, P. F. Knowles, and S. E. V. Phillips, Science 286, 1724 (1999); M. R. Parsons, M. A. Convery, C. M. Wilmot, K. D. S. Kapil, V. Blakeley, A. S. Corner, S. E. V. Phillips, M. J. McPherson, and P. F. Knowles, Structure 3,1171 (1995); J. M. Murray, C. G. Saysell, C. M. Wilmot, W. S. Tambyrajah, J. Jaeger, P. F. Knowles, S. E. V. Phillips, and M. J. McPherson, Biochemistry 38,8217 (1999)] (indicated by boxes) are available for species 1 (PDB code 1D6U), 4 (PDB code 1D6Z), and 5 (PDB code 1DYU).

Fig. 4. Catalytic intermediates in the oxidative half-reaction of E. coli amine oxidase. X-ray single-crystal structures [C. M. Wilmot, J. Hajdu, M. J. McPherson, P. F. Knowles, and S. E. V. Phillips, Science 286, 1724 (1999); M. R. Parsons, M. A. Convery, C. M. Wilmot, K. D. S. Kapil, V. Blakeley, A. S. Corner, S. E. V. Phillips, M. J. McPherson, and P. F. Knowles, Structure 3,1171 (1995); J. M. Murray, C. G. Saysell, C. M. Wilmot, W. S. Tambyrajah, J. Jaeger, P. F. Knowles, S. E. V. Phillips, and M. J. McPherson, Biochemistry 38,8217 (1999)] (indicated by boxes) are available for species 1 (PDB code 1D6U), 4 (PDB code 1D6Z), and 5 (PDB code 1DYU).

Crystals of Escherischia coli CuAO are grown under conditions that match the pH optimum for enzymatic activity (pH 7.2).23 The crystals are pale pink in color (peak at 480 nm), which is that expected for the TPQ chromophore, and

23 M. R. Parsons, M. A. Convery, C. M. Wilmot, K. D. S. Kapil, V. Blakeley, A. S. Corner, S. E. V. Phillips, M. J. McPherson, and P. F. Knowles, Structure 3,1171 (1995).

are rod-shaped (0.15 x 0.1 x 0.6 mm), making them ideal for crystal alignment on a single-crystal UV-visible microspectrophotometer to remove crystal prism effects. The crystal-stabilizing mother liquor contains 1.4 M sodium citrate and is relatively viscous, so the crystal is embedded in a Sephadex column to prevent it moving during kinetics.

The catalytic intermediate species for CuAOs have distinct spectroscopic features in the UV-visible range, based on protein and model studies. Using crystals of E. coli CuAO, the kinetics of the reaction with the substrate fi-phenylethy lamine were monitored by single-crystal UV-visible microspectrophotometry under aerobic conditions (Fig. 5). In solution, E. coli CuAO turns over /8-phenylethylamine at a rate of 118 molecules per second,24 but in the crystal the steady state is not reached for 8 min. A semiquinone species builds up in the crystal (twin peaks at 435 and 460 nm) before reaching a featureless steady state spectrum. The steady state could consist of a mixture of species, but in this case the spectrum was due to a single rate-determining species in the crystal, that of the iminoquinone, which has never been observed in solution, probably because it is transient. In the single-crystal X-ray structure of this rate-determining freeze-trapped intermediate, dioxygen can be seen bound to the enzyme, probably as the product, hydrogen peroxide.6 Proton transfer pathways are evident from the structure (species 4; Fig. 4): one by direct transfer from the 02 position of the quinone cofactor to dioxygen, and the other via the hydroxyl of a conserved tyrosine (Tyr-369 in E. coli CuAO) and a water (W2) from the 04 position of cofactor to dioxygen.

Unexpectedly, the product phenylacetaldehyde is observed at the back of the active site (Fig. 6). The exit channel for the product is formed at the interface of two protein domains. One of these domains is involved in a crystal contact, and this restricts the movement of one domain in relation to the other, thereby limiting "breathing" of the exit channel and making it more difficult for the product to exit the protein. The presence of the product explains why this species is rate determining in the crystal. The phenylacetaldehyde is keeping the catalytic base, Asp-383, protonated, and thereby preventing activation of a water (W4) that is present in the crystal structure for nucleophilic attack to release ammonia and regenerate fully oxidized TPQ (Fig. 6).

The semiquinone intermediate observed during turnover in the crystals, which could potentially be either species 2 or 3 in Fig. 4, can also be freeze trapped in liquid nitrogen. However, this species is sensitive to X-radiation at a wavelength of 0.9 A, reducing back to the aminoquinol form of the cofactor during data collection. This can be demonstrated by taking single-crystal spectra both before and after X-ray data collection, and shows the importance of always measuring a crystal spectrum after exposure to the X-ray beam.

24 J. M. Murray, C. G. Saysell, C. M. Wilmot, W. S. Tambyrajah, J. Jaeger, P. F. Knowles, S. E. V.

Phillips, and M. J. McPherson, Biochemistry 38, 8217 (1999).

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