Abstract
Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [Cu(M)(His)(2)(OH(2))(2)-Cu(H)(His)(3)(OH(2))] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [Cu(M)(His)(2)S(Met)-Cu(H)(His)(3)] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)-S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu-S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the Cu(M) Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the Cu(M) site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the Cu(M) center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.