The nature of O2 reactivity leading to topa quinone in the copper amine oxidase from Hansenula polymorpha and its relationship to catalytic turnover.

The copper amine oxidases (CAOs) catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain in the active site. Monooxygenation within the ring of tyrosine at a single Cu(II) site is unique in biology and occurs as an early step in the formation of TPQ. The mechanism of this reaction has been further examined in the CAO from Hansenula polymorpha (HPAO). When a Clark electrode fitted to a custom-made, gastight apparatus over a range of initial concentrations of O(2) was used, rates of O(2) consumption at levels greater than air are seen to be reduced relative to earlier results, yielding K(D)(apparent) = 216 microM for O(2). This is consistent with a mechanism in which O(2) binds reversibly to the active site, triggering a conformational change that promotes ligation of tyrosinate to Cu(II). The activated Cu(II)-tyrosinate species has been proposed to react with O(2) in a rate-limiting step, although it was also possible that breakdown of a putative peroxy-intermediate controlled TPQ formation. To test the latter hypothesis, Cu(II)-free HPAO was prepared with 3,5-ring-[(2)H(2)]-tyrosine incorporated throughout the primary sequence. The absence of an isotope effect on the rate of TPQ formation eliminates cleavage of this C-H bond in a proposed Cu(II)-aryl-peroxide intermediate as a rate limiting step. The role of methionine 634, previously found to moderate O(2) binding during the catalytic cycle, is shown here to serve a similar function in TPQ formation. As with catalysis, the rate of TPQ formation correlates with the volume of the hydrophobic side chain at position 634, implicating similar binding sites for O(2) during catalysis and cofactor biogenesis.