Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.     

A Ferritin from Dendrorhynchus zhejiangensis with Heavy Metals Detoxification Activity

Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd²⁺, Pb²⁺ and Fe²⁺. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd²⁺ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.     

Tim‐2 is the receptor for H‐ferritin on oligodendrocytes

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H‐ferritin and that H‐ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain‐containing protein‐2 (Tim‐2) was shown to bind and internalize H‐ferritin. In the present study we show that Tim‐2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H‐ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim‐2 expression. Application of a blocking antibody to the extracellular domain of Tim‐2 significantly reduces H‐ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim‐2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim‐2 is the H‐ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells.     

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta

Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two‐hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co‐immunoprecipitation. The pET‐28a‐FER147 prokaryotic expression vector was constructed. The expressed recombinant Fer147 was then isolated, purified, and refolded. When ferritins were treated by different heavy metals, several detection methods, including scanning electron microscopy (SEM), circular dichroism (CD), and inductively coupled plasma–mass spectrometry (ICP‐MS) were applied to examine the structures and functions of the new protein Fer147, recombinant P. esculenta ferritin (Rferritin), and natural horse‐spleen ferritin (Hferritin). SEM revealed that the three ferritin aggregates changed obviously after different heavy metals treatment, meanwhile, a little different in aggregates were detected when the ferritins were trapped by the same heavy metal. Hence, changes in aggregation structure of the three proteins are related to the nature of the different heavy metals and the interaction between the heavy metals and the three ferritins. CD data suggested that the secondary structure of the three ferritins hardly changed after different heavy metals were trapped. ICP–MS revealed that the ferritins exhibit different enrichment capacities for various heavy metals. In particular, the enrichment capacity of the recombinant Fer147 and Rferritin is much higher than that of hferritin.     

The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2–5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe²⁺ and phosphate. In the absence of ferritin, phosphate catalyzed Fe²⁺ oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10–20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe²⁺. Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe²⁺ and facilitate the transfer of the Fe³⁺ into apo transferrin in the presence of phosphate.     

ω‐Turn: A novel β‐turn mimic in globular proteins stabilized by main‐chain to side‐chain CH···O interaction

Mimicry of structural motifs is a common feature in proteins. The 10‐membered hydrogen‐bonded ring involving the main‐chain C?O in a β‐turn can be formed using a side‐chain carbonyl group leading to Asx‐turn. We show that the N? H component of hydrogen bond can be replaced by a C^γ‐H group in the side chain, culminating in a nonconventional C? H···O interaction. Because of its shape this β‐turn mimic is designated as ω‐turn, which is found to occur ～three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C? H···O interaction occurring between the terminal residues, constraining the torsion angles ?_{i + 1}, ψ_{i + 1}, ?_{i + 2} and χ′_{1(i + 2)} (using the interacting C^γ atom). Based on these angles there are two types of ω‐turns, each of which can be further divided into two groups. C^β‐branched side‐chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal‐binding sites. N‐linked glycosylation occurs at the consensus pattern Asn‐Xaa‐Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω‐turn, which may be the recognition site for protein modification. Location between two β‐strands is the most common occurrence in protein tertiary structure, and being generally exposed ω‐turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2015; 83:203–214. © 2014 Wiley Periodicals, Inc.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

Defining the metal specificity of a multifunctional biofilm adhesion protein

The accumulation associated protein (Aap) of Staphylococcus epidermidis mediates intercellular adhesion events necessary for biofilm growth. This process depends upon Zn²⁺‐induced self‐assembly of G5 domains within the B‐repeat region of the protein, forming anti‐parallel, intertwined protein “ropes” between cells. Pleomorphism in the Zn²⁺‐coordinating residues was observed in previously solved crystal structures, suggesting that the metal binding site might accommodate other transition metals and thereby support dimerization. By use of carefully selected buffer systems and a specialized approach to analyze sedimentation velocity analytical ultracentrifugation data, we were able to analyze low‐affinity metal binding events in solution. Our data show that both Zn²⁺ and Cu²⁺ support B‐repeat assembly, whereas Mn²⁺, Co²⁺, and Ni²⁺ bind to Aap but do not support self‐association. As the number of G5 domains are increased in longer B‐repeat constructs, the total concentration of metal required for dimerization decreases and the transition between monomer and dimer becomes more abrupt. These characteristics allow Aap to function as an environmental sensor that regulates biofilm formation in response to local concentrations of Zn²⁺ and Cu²⁺, both of which are implicated in immune cell activity.     

Ferritin blocks inhibitory effects of two-chain high molecular weight kininogen (HKa) on adhesion and survival signaling in endothelial cells

Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5β1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.     

Metal‐dependent assembly of a protein nano‐cage

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions were evaluated, with Ni²⁺ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.     

Competitive Adsorption of Copper‐Nickel and Copper‐Cadmium Binaries on SMW

In this study, the technical feasibility of using a low‐cost sorbent, sheep manure waste (SMW), for the removal of copper, nickel and cadmium ions from aqueous solutions containing Cu²⁺ ‐‐Cd²⁺ or Cu²⁺ ‐‐Ni²⁺ binaries is investigated. The scope of the study includes the investigation of the affinity of each metal ion in the presence of the other. Experimental results showed that the SMW has high affinity for the three studied heavy metals, and the affinity of the SMW for these metals is in the order copper > cadmium > nickel. The presence of copper in a solution containing Ni²⁺ or Cd²⁺ reduces significantly the percentage removal of both nickel and cadmium. On the other hand, the percentage removal of copper was not affected significantly by the presence of either nickel or cadmium. The equilibrium adsorption data were fit very well with Langmuir, Freundlich, Redlich‐Peterson, and Sips isotherm equations. The prediction of the binary adsorption from single metal adsorption data was in fair agreement with the experimental results. The role of ion exchange and carboxyl groups in the adsorption process were also studied.     

Design of novel ligands of CDP‐methylerythritol kinase by mimicking direct protein‐protein and solvent‐mediated interactions

The methylerythritol 4‐phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore‐directed virtual screening and a protein‐protein interaction (PPI)‐mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4‐(cytidine 5‐diphospho)‐2C‐methyl‐D‐erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water‐mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H‐furo[3,2‐g]chromen‐7‐one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology. Copyright © 2010 John Wiley & Sons, Ltd.     

A simply synthesized biphenyl substituted piperidin‐4‐one for the fluorescence chemosensing of Cd2+

Ion‐induced change in fluorescence is a straight‐forward method for detection of toxic metal ions showing immediate response. Cadmium ions are toxic to the environment. We report in this paper a piperidine‐4‐one‐based fluorescent chemosensor of Cd²⁺ ions, designed and synthesized by a simple method. The compound is characterized using infra‐red (IR) and ¹H–NMR spectral techniques. The chemosensor showed Cd²⁺ ion selectivity and sensitivity in aqueous solution. The stoichiometry and the binding constants were determined using fluorescence spectroscopy. Piperidine‐4‐one shows a 1:1 stoichiometric binding to Cd²⁺. The limit of detection of Cd²⁺ was reported.     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

Acidic microenvironment is commonly observed in ischaemic tissue. In the kidney, extracellular pH dropped from 7.4 to 6.5 within 10 minutes initiation of ischaemia. Acid‐sensing ion channels (ASICs) can be activated by pH drops from 7.4 to 7.0 or lower and permeates to Ca²⁺entrance. Thus, activation of ASIC1a can mediate the intracellular Ca²⁺ accumulation and play crucial roles in apoptosis of cells. However, the role of ASICs in renal ischaemic injury is unclear. The aim of the present study was to test the hypothesis that ischaemia increases renal epithelia cell apoptosis through ASIC1a‐mediated calcium entry. The results show that ASIC1a distributed in the proximal tubule with higher level in the renal tubule ischaemic injury both in vivo and in vitro. In vivo, Injection of ASIC1a inhibitor PcTx‐1 previous to ischaemia/reperfusion (I/R) operation attenuated renal ischaemic injury. In vitro, HK‐2 cells were pre‐treated with PcTx‐1 before hypoxia, the intracellular concentration of Ca²⁺, mitochondrial transmembrane potential (?ψm) and apoptosis was measured. Blocking ASIC1a attenuated I/R induced Ca²⁺ overflow, loss of ?ψm and apoptosis in HK‐2 cells. The results revealed that ASIC1a localized in the proximal tubular and contributed to I/R induced kidney injury. Consequently, targeting the ASIC1a may prove to be a novel strategy for AKI patients.     

Metal binding affinity and structural properties of calmodulin‐like protein 14 from Arabidopsis thaliana

In addition to the well‐known Ca²⁺ sensor calmodulin, plants possess many calmodulin‐like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca²⁺ and Mg²⁺ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS‐based fluorescence, to evaluate the structural effects of metal binding and metal‐induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca²⁺ ion with micromolar affinity (K_d ～ 12 µM) and the presence of 10 mM Mg²⁺ decreases the Ca²⁺ affinity by ～5‐fold. Although binding of Ca²⁺ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca²⁺ sensors, but causes only localized structural changes in the unique functional EF‐hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.     

Identification and characterization of novel ERC‐55 interacting proteins: Evidence for the existence of several ERC‐55 splicing variants; including the cytosolic ERC‐55‐C

ERC‐55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC‐55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC‐55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC‐55 splicing variants including ERC‐55‐C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub‐cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin‐6, kininogen and lysozyme with ERC‐55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca²⁺] of ～10^?7 M or greater, while calcyclin interaction requires [Ca²⁺] of >10^?5 M. Interaction with peroxiredoxin‐6 is independent of Ca²⁺. Co‐localization of lactoferrin, S100P and calcyclin with ERC‐55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC‐55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.     

Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E‐Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E‐Syt1 tethers and transports lipids in a Ca²⁺‐dependent manner, but the role of Ca²⁺ in this regulation is unclear. Of the five C2 domains of E‐Syt1, only C2A and C2C contain Ca²⁺‐binding sites. Using liposome‐based assays, we show that Ca²⁺ binding to C2C promotes E‐Syt1‐mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P₂‐rich membranes, as previously suggested by studies in semi‐permeabilized cells. Importantly, Ca²⁺ binding to C2A enables lipid transport by releasing a charge‐based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E‐Syt1 constructs defective in Ca²⁺ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E‐Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca²⁺‐triggered phosphatidylserine scrambling. Thus, a main effect of Ca²⁺ on E‐Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.     

Cupryphans,metal‐binding,redox‐active,redesigned conopeptides

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post‐translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well‐defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox‐active (macro)molecules was tested by engineering a copper‐binding site on two different variants of the natural peptide Contryphan‐Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg–Cupryphan, bind Cu²⁺ ions with a 1:1 stoichiometry and a K_d in the 100 nM range. Other divalent metals (e.g., Zn²⁺ and Mg²⁺) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.