Formation and Electronic Structure of an Atypical Cu_A Site

摘要

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 lambda(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 sigma(u)* ground state rather than the typical equilibrium between sigma(u)* and pi(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.