Assembling a unique three-copper center of thiocyanate dehydrogenase in vitro

Thiocyanate dehydrogenase (TcDH) plays a pivotal role in the decomposition of thiocyanate in some sulfur-oxidizing bacteria. Here we report the assembly mechanism of the unique three-copper center (Cu1, Cu2 and Cu3) of TcDH in vitro. Using EPR, we established the sequence of copper incorporation into the active center: copper ions are first incorporated in parallel into Cu1 and Cu2 sites, and finally into Cu3 site. TcDH shows high affinities for both Cu(II) (K_D values for three sites range from 2.0 × 10^-12 to 3.8 × 10^-17 M) and Cu(I) (average K_D values of three sites are 1.0 × 10^-13 M). The process of assembling the TcDH copper center, and thus acquiring the enzyme catalytic activity, is kinetically fast with Cu(I) (~min) ions but extremely slow with Cu(II) (~hours), which correlates with the rate of Cu(II) incorporation into the protein. The inactivation of TcDH due to removal of copper from the active site is kinetically slow due to the low accessibility of the copper center. This phenomenon appears to act as an adaptation mechanism that prevents unwanted loss of copper in vivo due to interaction with the higher-affinity copper binders. Based on the slow incorporation of Cu(II) into the TcDH active center in vitro, we suggest that it is Cu(I) that is involved in the process of assembling of TcDH copper center in vivo.