The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

John, Kenneth R.; Lee, Sheeyong; Choi, Moonsung; Bishop, G. Reid; Hosler, Jonathan P.; Davidson, Victor L.

doi:10.1016/j.jinorgbio.2007.09.007

Cited by 7 publications

(12 citation statements)

References 15 publications

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“…This was in part due to the presence of zinc (24). An additional procedure (25) was required to unfold the protein, remove any metal ions, and incorporate Cu 2+ into the refolded protein.…”

Section: Methodsmentioning

confidence: 99%

Defining the Role of the Axial Ligand of the Type 1 Copper Site in Amicyanin by Replacement of Methionine with Leucine

et al. 2009

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The effects on the structure and function of amicyanin of replacing the axial methionine ligand of the type 1 copper site with leucine have been characterized. The crystal structures of the oxidized and reduced forms of the protein reveal that the copper site is now tri-coordinate with no axial ligand, and that the copper coordination distances for the two ligands provided by histidines are significantly increased. Despite these structural changes, the absorption and EPR spectra of M98L amicyanin are only slightly altered and still consistent with that of a typical type 1 site. The oxidation-reduction midpoint potential (E m ) value becomes 127 mV more positive as a consequence of the M98L mutation, most likely due to increased hydrophobicity of the copper site. The most dramatic effect of the mutation was on the electron transfer (ET) reaction from reduced M98L amicyanin to cytochrome c-551i within the protein ET complex. The rate decreased 435-fold, which was much more than expected from the change in E m value. Examination of the temperature dependence of the ET rate (k ET ) revealed that the mutation caused a 13.6 fold decrease in the electronic coupling (H AB ) for the reaction. A similar decrease was predicted from a comparative analysis of the crystal structures of reduced M98L and native amicyanins. The most direct route of ET for this reaction is through the Met98 ligand. Inspection of the structures suggests that the major determinant of the large decrease in the experimentally determined values of H AB and k ET is the increased distance from the copper to the protein within the type 1 site of M98L amicyanin.Copper proteins are common in nature and involved in a variety of biological processes such as respiration, photosynthesis, and redox reactions critical to metabolism (1). The copper centers are classified according to their spectroscopic properties as type 1, type 2 or type 3. Type 1 copper sites are found in a wide range of electron transfer (ET) proteins, including amicyanin and azurin in bacteria, plastocyanin in plants, and multicopper oxidases such as NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 October 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript fungal laccase and human ceruloplasmin (1). The type 1 copper site of cupredoxins consists of three relatively strong equatorial ligands, nitrogens of two histidines and sulfur of a cysteine, forming a trigonal plane. A weaker fourth axial ligand is usually, but not always, provided by a sulfur of a methionine (2). Azurin is atypical in that additional coordination to the copper is provided by the backbone carbonyl O of a Gly, giving that copper site a trigonal bipyramidal geometry. In amicyanin from P. denitrificans the three strong equatorial copper ligands are provided by residues His53, His95 and Cys92, and the weak axial ligand is provided by Met98 (3).Amicyanin (4) serves as a mediator of ET from methylamine dehydrogenase (MADH) (5) to cytochrome c-551i (6)....

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“…This was in part due to the presence of zinc (24). An additional procedure (25) was required to unfold the protein, remove any metal ions, and incorporate Cu 2+ into the refolded protein.…”

Section: Methodsmentioning

confidence: 99%

Defining the Role of the Axial Ligand of the Type 1 Copper Site in Amicyanin by Replacement of Methionine with Leucine

et al. 2009

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“…It was previously observed that the as-isolated M98A, M98Q and M98L amicyanins from P. denitrificans did not have full occupancy of copper in the type 1 site [13, 14]. Instead there was partial occupancy of the metal site by zinc rather than copper.…”

Section: Resultsmentioning

confidence: 99%

“…However, previously generated Met98 mutants of amicyanin were isolated with the site at least partially occupied by zinc. It was demonstrated that the influence of the axial ligand of the type 1 copper site on metal specificity for incorporation of copper or zinc into amicyanin is strongest prior to the completion of protein folding and adoption of the final type 1 site geometry [13]. However, in all other Met98 amicyanin mutants it was possible to remove the bound zinc and reconstitute the protein with copper.…”

Section: Discussionmentioning

confidence: 99%

“…A comparative study of the specificity of the type 1 site of native and M98Q amicyanins for either Cu 2+ or Zn 2+ revealed that the influence of the axial ligand on metal specificity is strongest prior to the completion of protein folding and adoption of the final type I site geometry [13]. In this study, the role of the axial ligand in determining the metal specificity during and after protein folding was further examined by mutation of Met98 of amicyanin to Lys.…”

Section: Introductionmentioning

confidence: 97%

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Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper

Sukumar

Choi

Davidson

2011

Journal of Inorganic Biochemistry

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The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn2+ at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn2+. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.

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“…The mauC gene encodes amicyanin, a Type I copper protein that is localized in the periplasm [11]. It was shown that incorporation of the very tightly bound copper occurs during the folding of amicyanin in the periplasm [25]. In cases such as this, the timing of the translocation of the protein is important to avoid mis-folding and aggregation that can result from unavailability of copper at the critical point in protein folding.…”

Section: Discussionmentioning

confidence: 99%

Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain

Dow

Tatulian

Davidson

2015

Protein Expression and Purification

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Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans, was used to express the light chain variable domain of the human κIO8/O18 germline antibody in the periplasm of E. coli. The expressed protein was purified in good yield (70 mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag. Circular dichroism spectroscopy was used to determine if the secondary and tertiary structures of the protein and its thermal stability corresponded to those of the native folded protein. The expressed and purified protein was indeed properly folded and exhibited a reasonable thermal transition temperature of 53°C. These results indicate that the amicyanin signal sequence may be particularly useful for prokaryotic expression of proteins which are prone to mis-folding, aggregation or formation of inclusion bodies, all of which were circumvented in this study.

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The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

Cited by 7 publications

References 15 publications

Defining the Role of the Axial Ligand of the Type 1 Copper Site in Amicyanin by Replacement of Methionine with Leucine

Defining the Role of the Axial Ligand of the Type 1 Copper Site in Amicyanin by Replacement of Methionine with Leucine

Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper

Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain

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