Open AccessA Binuclear CuA Center Designed in an All α-Helical Protein Scaffold
Author(s) -
Evan N. Mirts,
Sergei A. Dikanov,
Anex Jose,
Edward I. Solomon,
Yi Lu
Publication year - 2020
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/jacs.0c04226
Subject(s) - chemistry , methane monooxygenase , delocalized electron , electron paramagnetic resonance , electron transfer , magnetic circular dichroism , redox , crystallography , metalloprotein , copper protein , valence (chemistry) , circular dichroism , photochemistry , nuclear magnetic resonance , metal , inorganic chemistry , copper , spectral line , biochemistry , physics , organic chemistry , astronomy , catalysis
The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear Cu A center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of Cu A are difficult to reproduce in synthetic models, and every artificial protein Cu A center to-date has used a modified cupredoxin. Here, we present a fully functional Cu A center designed in a structurally nonhomologous protein, cytochrome c peroxidase (C c P), by only two mutations (Cu A C c P). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that Cu A C c P is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact g z area and small A z hyperfine principal value with g and A tensors that resemble axially perturbed Cu A . Stopped-flow kinetics found that Cu A formation proceeds through a single T2Cu intermediate. The reduction potential of Cu A C c P is comparable to native Cu A and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the C c P heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.