Binuclear Cu_A formation in biosynthetic models of Cu_A in azurin proceeds via a novel Cu(Cys)₂His mononuclear copper intermediate

Journal Article · Wed Sep 09 00:00:00 EDT 2015 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.5b00659· OSTI ID:1344903

Chakraborty, Saumen ^[1]; Polen, Michael J. ^[1]; Chacon, Kelly N. ^[2]; Wilson, Tiffany D. ^[1]; Yu, Yang ^[1]; Reed, Julian ^[1]; Nilges, Mark J. ^[1]; Blackburn, Ninian J. ^[2]; Lu, Yi ^[1]

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1. Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)
2. Oregon Health & Sciences Univ., Portland, OR (United States)

Cu_A 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 Cu_A centers facilitate efficient ET (k_ET > 10⁴ s^–1) under low thermodynamic driving forces (10–90 mV). While the structure and functional properties of Cu_A are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu_A maturation process are still lacking. Previous studies of the Cu_A assembly mechanism in vitro using a biosynthetic model Cu_A center in azurin (Cu_AAz) identified a novel intermediate X (I_x) during reconstitution of the binuclear site. However, because of the instability of I_x and the coexistence of other Cu centers, such as Cu_A' and type 1 copper centers, the identity of this intermediate could not be established. In this paper, we report the mechanism of Cu_A assembly using variants of Glu114XCu_AAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu_A site, with a major focus on characterization of the novel intermediate I_x. We show that Cu_A assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate I_x, 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 → I_x formation, suggesting that Cu_A 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 I_x 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 I_x as a mononuclear Cu(Cys)₂(His) complex. Finally, because some of the intermediates have been proposed to be involved in the assembly of native Cu_A, these results shed light on the structural features of the important intermediates and mechanism of Cu_A formation.

• Research Organization: Univ. of Illinois at Urbana-Champaign, IL (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES)
• Grant/Contract Number: AC02-98CH10886
• OSTI ID: 1344903
• Journal Information: Biochemistry, Vol. 54, Issue 39; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY