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Formation and Electronic Structure of an Atypical CuA Site

Ross, Matthew O., Fisher, Oriana S., Morgada, Marcos N., Krzyaniak, Matthew D., Wasielewski, Michael R., Vila, Alejandro J., Hoffman, Brian M., Rosenzweig, Amy C.
Journal of the American Chemical Society 2019 v.141 no.11 pp. 4678-4686
copper, cysteine, electron paramagnetic resonance spectroscopy, geometry, histidine, ligands, methanotrophs, nuclear magnetic resonance spectroscopy, proteins
PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper CuA center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD CuA bear similarities to those of canonical CuA sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric CuA site to two mononuclear Cu²⁺ sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD CuA formation occurs rapidly and involves a short-lived intermediate with a λₘₐₓ of 360 nm. Unlike other CuA sites, the PmoD CuA is unstable, decaying to two type 2 Cu²⁺ centers. Surprisingly, NMR data indicate that the PmoD CuA has a pure σᵤ* ground state rather than the typical equilibrium between σᵤ* and πᵤ of all other CuA proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the CuA core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known CuA sites.