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

Abstract

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)....

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

“…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.…”

“…M98K amicyanin was subjected to a procedure which was previously developed to remove Zn 2+ from other amicyanin mutants and then to reconstitute those proteins with Cu 2+ [14, 17]. To completely unfold the protein and remove any metal ions, M98K amicyanin was incubated in 10 mM HEPES buffer, pH 8.0, containing 6 M guandinine-HCl, 50 mM EDTA and 2 mM dithiothreitol.…”

“…Met98 of P. denitrificans amicyanin was previously mutated to Ala, Gln, and Leu [11–14]. These studies indicated that the position and rigidity of the axial ligand of the type 1 copper site influences the overall protein stability, the active site ligation geometry, the spectroscopic properties, and ET properties.…”

Theoretical study on the degree of delocalization of unpaired spin in two mixed valence copper(II/I) complexes with isomeric chelating diamines and iodide

“…The chemistry of mixed valence copper(II)-copper(I) complexes become very interesting now-adays due to their potential ability to mimic the active site structures of several metalloenzymes and metalloproteins [1,2]. In addition, copper(I) complexes show interesting photochemical and photophysical properties, with potential applications as light-emitting diodes, luminescent probes, and photovoltaics [3][4][5][6][7].…”

The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site

“…Amicyanin from Paracoccus denitrificans [4, 5] is a cupredoxin which mediates electron transfer from methylamine dehydrogenase (MADH) [6] to cytochrome c -551i [7]. Crystal structures of amicyanin alone [8] and in complex with its redox partner proteins [9, 10] have been determined, and spectroscopic, redox, kinetic and site-directed mutagenesis studies have described structure-function relationships that define the roles of specific amino acid residues of amicyanin in recognition of redox partners and mediation of interprotein electron transfer [11–17]. …”