Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

“…As reported previously (29,40,53), rapid spectral changes associated with changes in the redox and chemical state of TPQ (TPQ ox 3 TPQ ssb 3 TPQ psb 3 TPQ amr 3 TPQ sq ) were observed (Fig. 4).…”

“…Stopped-flow Measurements-Transient kinetic analyses were done at 4°C with an Applied Photophysics stopped-flow spectrophotometer (40,41). Typically, equal volumes (about 30 l each) of enzyme (200 M monomer in 50 mM HEPES buffer, pH 6.8) and substrate (1 mM 2-PEA) solutions were mixed in a 20-l mixing cell by triggering with an N 2 gas piston; the mixing dead time was generally 2.3 ms at an N 2 gas pressure of 500 kilopascals.…”

“…is well established that the absorption spectrum of the bound cofactor TPQ changes rapidly during the reductive half-reaction of CAOs (1)(2)(3), as demonstrated by stopped-flow spectrophotometry of the reaction with AGAO (29,40,53). The final form of TPQ in the reductive half-reaction without turnover (measured under anaerobic conditions) is TPQ sq , which exhibits characteristic absorption maxima at about 440 and 470 nm with a shoulder at about 350 nm.…”

“…Before x-ray analysis, these crystals were subjected to single-crystal microspec- trophotometry to identify the reaction intermediates present within them (29,40,41). The absorption spectra of the frozen crystals anaerobically soaked with 4 mM 2-PEA or 10 mM histamine (these crystals were designated AGAO PEA and AGAO HTA , respectively) (Fig.…”

“…We have previously determined x-ray crystal structures of the intermediates, including TPQ ssb and TPQ psb , formed during the reductive half-reaction of AGAO with 2-phenylethylamine (2-PEA) (29), tyramine (40), and ethylamine (41). In each case, the TPQ cofactor in these structures had an off-copper conformation.…”