Binuclear Cu_A Formation in Biosynthetic Models of Cu_A in Azurin Proceeds via a Novel Cu(Cys)₂His Mononuclear Copper Intermediate

Abstract

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 copper incorporation 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, due to 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, 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 the CuA formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggests that the nature of the Glu114 side chain influences the timescales 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 mechanism and optimization of the conditions, we have obtained Ix in ~80–85% population in these variants, which allowed us to employ UV-Vis, EPR, and EXAFS spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)2(His) complex. Since 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.

Understanding Factors that Control the Structural (Dis)Assembly of Sulphur-Bridged Bimetallic Sites

“…More precisely, the energy that is required for two S-Ni-L fragments to adopt to the transition state geometry is expected to benefit from diverging stereoelectronic properties of the nickel ions. This proposal explains the marked kinetic preference of formation of sulphur-bridged [Ni 2 (µ-S) 2 ] + , Ni = Ni(I)/Ni(II), from geometrically independent, heterovalent nickel sites, and augments mechanistic data reported for the skeletal assembly of Cu A [1, [6][7][8][9]. This mechanism may also apply to the self-assembly of the mixed-valent form of 2Fe-ferredoxins, for which, however, experimental…”

“…Whereas the understanding of the relationship between biological function and electronic properties of closed-and open-shell forms of Cu A and 2Fe-ferredoxins has been widely developed, details on the mechanism of formation and of the parameters that control structural persistence of [M 2 (µ-S) 2 ] sites have remained largely unknown. In vitro studies starting from an apoprotein concluded that Cu A assembles rapidly in form of its open-shell state after stepwise introduction of Cu(I) and Cu(II) sites [6][7][8][9]. Different from natural Cu A , preparative assembly of the bimetallic core [Cu 2 (µ-S) 2 ] commonly involves dimerization of homovalent precurors [10][11][12][13].…”

Copper(I) Thiolate Heteroadamantane Cage Structures with Relevance to Metalloproteins

“…Although the structure of Cu A and its biological functions are well‐known, the incorporation of copper in the proteins and, in particular, the formation of the mixed‐valent dinuclear centre is one of the remaining challenges in understanding Cu A . This assembly proceeds through several different types of mononuclear copper centres, and a new intermediate Cu(Cys) 2 His complex has been identified recently …”

Mechanistic Aspects of Redox‐Induced Assembly and Disassembly of S‐Bridged [2M‐2S] Structures

“…Their propensity to self‐assemble spontaneously has not only been utilized to prepare [2M‐2S] structures of Fe and Cu synthetically from mononuclear precursors but also appears to play a role in biosynthesis . Quantitative insight into this process is limited to the in vivo/vitro maturation of the Cu A [2Cu‐2S] + site either in an apoprotein or models thereof –. Notably, one electron reduction of [2Fe‐2S] 2+ cores triggers self‐assembly into [4Fe 2.5+ ‐4S] 2+ .…”