Formation and Electronic Structure of an Atypical Cu Site.

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper Cu center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD Cu bear similarities to those of canonical Cu sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric Cu site to two mononuclear Cu sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD Cu formation occurs rapidly and involves a short-lived intermediate with a λ of 360 nm. Unlike other Cu sites, the PmoD Cu is unstable, decaying to two type 2 Cu centers. Surprisingly, NMR data indicate that the PmoD Cu has a pure σ* ground state rather than the typical equilibrium between σ* and π of all other Cu proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the Cu core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known Cu sites.