Formation and Electronic Structure of an Atypical Cu_A Site

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper Cu_A center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD Cu_A bear similarities to those of canonical Cu_A sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric Cu_A site to two mononuclear Cu²⁺ sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD Cu_A formation occurs rapidly and involves a short-lived intermediate with a λ_max of 360 nm. Unlike other Cu_A sites, the PmoD Cu_A is unstable, decaying to two type 2 Cu²⁺ centers. Surprisingly, NMR data indicate that the PmoD Cu_A has a pure σ_u* ground state rather than the typical equilibrium between σ_u* and π_u of all other Cu_A proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the Cu_A core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known Cu_A sites.