CuA is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N2ORs), and nitric oxide reductase (NOR). In these proteins, the CuA centers facilitate efficient ET (kET > 104 s-1) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of CuA are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the CuA maturation process are still lacking. Previous studies of the CuA assembly mechanism in vitro using a biosynthetic model CuA center in azurin (CuAAz) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as CuA′ and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of CuA assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the CuA site, with a major focus on characterization of the novel intermediate Ix. We show that CuA assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that CuA formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)2(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native CuA, these results shed light on the structural features of the important intermediates and mechanism of CuA formation.
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