A New Type 2 Copper Cysteinate Azurin

Amsterdam, Irene M. C. van; Ubbink, Marcellus; Bosch, M.W.; Rotsaert, Frederik A.J.; Sanders-Loehr, Joann; Canters, Gerard W.

doi:10.1074/jbc.m202977200

Cited by 20 publications

(20 citation statements)

References 55 publications

(75 reference statements)

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“…Complex 1 has an intense electronic absorption at 380 nm (ε, 3245 M −1 cm −1 ) corresponding to a monothiolate, RS − → Cu 2+ CT transition and a d-d absorption centered at 640 nm (ε, 540 M −1 cm −1 ). These absorptions are indicative of a type-2 tetragonally distorted copper site as observed earlier with various enzymatic and synthetic copper-monothiolate systems 2,4,39–42. HPLC of 1 shows clearly that the yellow species is a monomeric form of the protein.…”

Section: Resultssupporting

confidence: 73%

“…For C49A, the situation is more complex, since analogy to the H117/N42C variant suggests that the 380 nm absorption band could be assigned to more than one yellow species 39. Indeed, the EPR data detect two distinct Cu(II) species with different electronic properties, and although EXAFS simulations appear to support a similar ligand set to C45A, the presence of multiple speciation makes it harder to draw firm conclusions.…”

Section: Disscussionmentioning

confidence: 99%

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

et al. 2010

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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.

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Section: Resultssupporting

confidence: 73%

Section: Disscussionmentioning

confidence: 99%

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

et al. 2010

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“…It is also possible that the Fe II -sulfur interactions are weak and not detectable using this method due to concentration limitations of the experiments. That 3-carboxypropyl disulfide inhibits ironbut not copper-mediated DNA damage is an unexpected result, since copper readily binds methionine and cysteine in vivo [74,75]. Thus, experimental evidence that antioxidant-iron coordination is required for DNA damage inhibition is ambiguous, and another mechanism may be partially responsible for the observed activity.…”

Section: The Role Of Metal Coordination In Antioxidant Activitymentioning

confidence: 96%

Metal specificity in DNA damage prevention by sulfur antioxidants

Battin

Brumaghim

2008

Journal of Inorganic Biochemistry

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“…The rR spectrum shows vibrational features at 257 and 343 cm −1 , assigned to Cu-S stretches (Figure 111D). 645,646 Based on these vibrations and the lack of Tyr ring modes, the 406 nm band has been assigned as a Cys to Cu(II) charge transfer transition. At longer incubation times (24 h), the bleached species is formed.…”

Section: Copper Active Sites That Activate Dioxygenmentioning

confidence: 99%