The Type 1 Blue Copper Site: From Electron Transfer to Biological Function.

Cupredoxins host in their scaffold one of the most studied and interesting metal sites in biology: the type 1 (T1) or blue Cu center. Blue Cu proteins have evolved to play key roles in biological electron transfer and have the ability to react with a wide variety of redox partners. The inner coordination sphere of T1 Cu sites conserves two histidines and one cysteine with a short Cu-S(Cys) bond as ligands in a trigonal arrangement, with a variable axial ligand that modulates the electronic structure and reactivity. The structural, electronic and geometric features of T1 Cu centers provide the basis for a site that can be optimized by the protein structure for each biological function. This chapter highlights the properties that make this unique Cu center in biology an efficient and tunable electron transfer site. The contributions of the first coordination shell and the high covalency of the Cu-S(Cys) bond in the T1 Cu site to its distinctive geometric and spectroscopic features are discussed, as well as the role of the protein scaffold in imposing an 'entatic' state with a distorted tetrahedral geometry that minimizes geometric changes upon redox cycling. The analysis of naturally occurring perturbed blue Cu sites provides further insights into how the protein scaffold can tune the properties of the T1 Cu site. Blue Cu sites display a wide range of reduction potentials, as these are tuned to be consistent with their physiologically relevant electron donors and acceptors. The different properties of the protein matrix that play important roles in finetuning the reduction potential of T1 Cu sites are also discussed, including the nature of the axial ligand and outer coordination sphere effects. These concepts are further illustrated by the discussion of examples of biosynthetic blue Cu proteins. Finally, the different features of the T1 Cu site that make it an optimal site for electron transfer (ET) are discussed, in terms of Markus theory for intra- and inter-molecular ET. The active site in multicopper oxidases is used as an example to illustrate the contributions of the anisotropic covalency of the blue Cu site to an efficient ET, while the diverse reactivity of the T1 Cu sites in these enzymes is discussed to dissect the different properties provided by the protein that help tune these unique sites for biological ET.

Effects of alpha-synuclein post-translational modifications on metal binding.

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.

Spectroscopic and Theoretical Study of Cu(I) Binding to His111 in the Human Prion Protein Fragment 106-115.

The ability of the cellular prion protein (PrP(C)) to bind copper in vivo points to a physiological role for PrP(C) in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrP(C). Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting Cu(I) and Cu(II) binding properties. We have evaluated Cu(I) coordination to the PrP(106-115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. Cu(I) coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5-8, both methionine (Met) residues bind to Cu(I), forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrP(C) (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrP(C) to maintain the bound Cu(I) ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant Cu(I)-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrP(C). This study provides further insight into the Cu(I) coordination properties of His111 in human PrP(C) and the molecular mechanism of oxygen activation by this site.

Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells.

Levels of amyloid beta (Aß) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aß. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aß in differentiated SH-SY5Y cells incubated with 1-20 µM of Hg. Exposure to 20 µM of Hg induced an increase in Aß-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aß-42 degradation. Finally, the comparative effects of lead (Pb, 50 µM) were evaluated. We found a significant increase in Aß-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aß levels by different mechanisms.