Chemical issues addressing the construction of the distal Ni[cysteine-glycine-cysteine]2- site of acetyl CoA synthase: why not copper?

Abstract

The discovery of the metallopeptide Ni(Cysteine-Glycine-Cysteine)(2-), Ni(CGC)(2-), in the A-cluster active site of Acetyl CoA Synthase has prompted the synthesis of many small molecule models which employ M(N(2)S(2)) complexes as metalloligands. In vitro studies have shown that nickel incorporates into the N(2)S(2) binding pocket even when copper is in the enzyme growth medium, while copper is preferentially taken up in the proximal site, displacing the catalytically active nickel. (Darnault, C.; Volbeda, A.; Kim, E.J.; Legrand, P.; Vernede, X.; Lindahl, P.A.; Fontecilla-Camps, J.C. Nat. Struct. Biol. 2003, 10, 271-279.) The work herein has been designed to address the chemical viability of copper(II) within the tripeptide N(2)S(2) ligand set. To this end, a series of CuN(2)S(2)(2-) complexes, the resin-bound, O-Cu(CGC)(2-) (A) and free Cu(CGC)(2-) (B) complexes, as well as Cu(ema)(2-) (C) and Cu(emi)(2-) (D) dianions, have been characterized by UV-vis, electron paramagnetic resonance (EPR), and electrospray ionization mass spectrometry (ESI-MS) spectroscopies, cyclic voltammetry (CV), and, where appropriate, X-ray diffraction studies, and compared to the Ni(II) congeners. EPR spectroscopic results have indicated that, in frozen N,N-dimethylformamide (DMF) solution, the copper complexes are distorted square planar structures with nitrogen and sulfur donors. This is consistent with X-ray diffraction measurements which also show copper(II) in a distorted square planar environment that is bereft of CuN(2)S(2)(2-) intermolecular interactions. Density-functional theory (DFT) calculations resulted in optimized structures that are consistent with crystallographic data and indicated highest occupied molecular orbital (HOMO)-singly occupied molecular orbital (SOMO) gaps of 5.01 and 4.68 eV for C and D, respectively. Optimized structures of Ni(ema)(2-) and Ni(emi)(2-) share the same basic characteristics as the copper(II) congeners. Electrochemical characterization of C and D resulted in a reversible Cu(III/II) couple at -1.20 V and - 1.40 V, respectively. Reactivity studies with Rh(CO)(2)(+) show similar donor capabilities for complexes A-D. Analysis of A shows that transmetalation does not occur. From competitive metal uptake studies on immobilized tripeptide it is concluded that the N(2)S(2)(4-) ligating unit has a slight preference for Cu(2+) over Ni(2+) and that the biosynthetic pathway responsible for constructing the distal site of ACS must be selective for nickel insertion or copper exclusion, or both.

Figure 1

Acetyl CoA Synthase: Representation of the A-Cluster (Ni_d,p = distal and proximal relative to the 4Fe4S cluster).,

Figure 2

Examples of synthetic monomer Cu^IIN₂S₂ and CuN₂SS' complexes: Cu(Cys-Et-Cys) = [Dimethyl N,N'-ethylenebis(L-cysteinato)-S,S']copper(II), [Cu(2-HL^N₂S)SR] = 2-methylthio-N-(2-pydridylmethyl)acetamide copper (II); Cu(phmi)^2- = N,N'-1,2-phenylenebis(2-mercapto-2-methylpropionamide)copper(II).,

Chart 1

Figure 3

Crystal packing diagram of [Et₄N]₂[Cu(ema)]•2H₂O showing H-Bonding and distances (Å) between CuN₂S₂ ^2- molecules.

Figure 4

Examples of metal carbonyl derivatives derived from NiN₂S₂ ^x- complexes: E = Ni(bme-daco)W(CO)₄, F = Ni(bme-daco)Rh(CO)₂ ⁺, G = [Ni(ema)Rh(CO)₂]₂ ^2-.

Figure 5

Frontier molecular orbitals of Ni(ema)^2-,, Ni(emi)^2-, Cu(ema)^2-, and Cu(emi)². For the nickel complexes, the orbitals descend in the order LUMO, HOMO, and HOMO-1. For the copper complexes, the orbitals descend in the order SOMO, HOMO, and HOMO-1.

Figure 6

(From right to left) Electrostatic potential maps of Ni(ema)^2-, Ni(emi)^2-, Cu(ema)^2-, and Cu(emi)^2- plotted at an isosurface value of 0.01. The range was taken from 1.00 (the most electropositive region, dark blue) to 0.33 (the most electronegative region, bright red). The view is oriented from the top onto the MN₂S₂ plane.

Figure 7

CV overlay of the Ni^III/II and Cu^III/II couples of [Et₄N]₂[Ni(ema)] (3.7 mM) (—), [Et₄N]₂[Ni(emi)] (4.2 mM) (···), [Et₄N]₂[Cu(ema)] (4.0 mM) (---),[Et₄N]₂[Cu(emi)] (2.3 mM) (—) using N,N'-dimethylformamide solvent at a scan rate of 100 mV/s, a Ag/AgNO₃ reference electrode, 0.1 M [ⁿBu₄N][BF₄] electrolyte, and a glassy-carbon electrode.

Figure 8

Schematic of the resin bound O-CGC/M²⁺ loaded beads. (S = solvent molecules, Rink linker = trialkoxybenzhydrylamine)

Figure 9

X-Band EPR spectra of (a) O-Cu(CGC)^2-, Complex A; (b) O-CGC incubated with 5 equiv of Cu(OAc)₂; (c) O-NH₂ incubated with Cu(OAc)₂. (Spectra are obtained from dried beads packed into an EPR tube at 9K and 9.75 GHz.) (O = NovaSyn TGR Beads)