Anatomy of a Red Copper Center: Spectroscopic Identification and Reactivity of the Copper Centers of Bacillus subtilis Sco and Its Cys-to-Ala Variants

Abstract

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the WT protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/vis, Cu and S K edge XAS, EPR, and resonance Raman spectroscopy. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form which is shown by EXAFS to be composed of a novel 2-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49 H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans which facilitate the formation of the highly stable 2-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at ~ 2.24 Å corresponding to the two thiolate copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at ~ 2.16 Å. S K-edge data gives a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, whence it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron transfer reactivity.

Electronic Structure of a CuII–Alkoxide Complex Modeling Intermediates in Copper-Catalyzed Alcohol Oxidations

“…38–40 The five-coordinate thiolate-bound green and red copper sites in nitrite reductase, nitrosocyanin, and BSco exhibit multiple Cu-S frequencies between 340-450 cm −1 , 300-350 cm −1 , and 310-380 cm −1 respectively. 26,28,41 Small molecules with similar pyrazolyl ligand scaffolds typically exhibit slightly simpler resonance Raman patterns. A type 1 blue copper mimetic molecule (HB(3,5-ipr 2 pz) 3 )Cu-SC(CH 3 ) 3 exhibits three frequencies at 348, 400, and 437 cm −1 assigned as δ(C-C-S), δ(C-C-C), and ν(Cu-S), respectively.…”

“…In addition, the higher energy S(Cys) p σ → Cu II d x 2 – y 2 transition is more intense, indicating that the lobes of the Cu II d x 2 – y 2 orbital lie along the Cu-S bond and that the bonding interaction is dominantly σ in character. 26,28 These five-coordinate red copper proteins generally have type 2 copper EPR spectra (large Cu A zz > 100 · 10 −4 cm −1 and g zz ≈ 2.2 > g yy ≈ g xx ) also indicative of a d x 2 – y 2 ground state. Although Cu II -O(TFE) shares the red color of nitrosocyanin and BSco, it is four-coordinate and the alkoxide ligand exhibits both π and σ bonding.…”

The Lumenal Loop Met672–Pro707 of Copper-transporting ATPase ATP7A Binds Metals and Facilitates Copper Release from the Intramembrane Sites

“…Given that EXAFS fitting cannot determine shell occupancies to better than ϳ25% precision, we also analyzed bond lengths, which are good indicators of coordination number. For both copper binding stoichiometries, the Cu(I)-N(His) bond length was 1.88 Ϯ 0.2 Å, a value typical of linear 2-coordination in either Cu(I)N 2 (22, 23) or mixed Cu(I)NS environments (24). The Cu(I)-S distance was 2.23 Ϯ 0.2 Å, a value closer to the typical 3-coordinate distance of 2.25-2.30 Å (17,18,(25)(26)(27).…”

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

“…The latter inference stems from the fact that Sco1 possesses the thioredoxin fold. 50, 54, 55 In Bacillus subtilis, another protein, YpmQ, which is the yeast homologue of Sco1, and has a Cu(Cys) 2 His ligation, has also been proposed to be required for Cu A assembly. Furthermore, the periplasmic protein PCu A C, found in several prokaryotes has been shown to deliver Cu(I) to apo Cu A , forming the binuclear Cu(I)-Cu A site.…”