Transformation of the Cu<sub>A</sub> Redox Site in Cytochrome <i>c</i> Oxidase into a Mononuclear Copper Center

Zickermann, Volker; Wittershagen, Axel; Kolbesen, Bernd O.; Ludwig, Bernd

doi:10.1021/bi962040e

Cited by 37 publications

(36 citation statements)

References 26 publications

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“…This is in contrast to site-directed mutants of Cu A active sites, which result in blue, red, or otherwise altered proteins as expected when the active-site electronic structure is perturbed (43)(44)(45)(46)(47)(48). CcO truncates exhibiting transient color changes before forming the thermodynamically stable Cu A site have been reported but, to our knowledge, have not been investigated further (41).…”

Section: Discussionmentioning

confidence: 81%

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Savelieff¹,

Wilson²,

Youssef³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The cupredoxin fold is an important motif in numerous proteins that are central to several critical cellular processes ranging from aerobic and anaerobic respiration to catalysis and iron homeostasis. Three types of copper sites have been found to date within cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, and purple Cu A. Although as much as 90% sequence difference has been observed among some members of this superfamily of proteins that span several kingdoms, sequence alignment and phylogenic trees strongly suggest an evolutionary link and common ancestry. However, experimental evidence for such a link has been lacking. We report herein the observation of pH-dependent transformation between blue T1 copper, red T2 copper, and the native purple Cu A centers of nitrous oxide reductase (N2OR) from Paracoccus denitrificans. The blue and red copper centers form initially before they are transformed into purple Cu A center. This transformation process is pH-dependent, with lower pH resulting in fewer trapped T1 and T2 coppers and faster transition to purple Cu A. These observations suggest that the purple CuA site contains the essential elements of T1 and T2 copper centers and that the Cu A center is preferentially formed at low pH. Therefore, this work provides an underlying link between the various cupredoxin copper sites and possible experimental evidence in vitro for the evolutionary relationship between the cupredoxin proteins. The findings also lend physiological relevance to cupredoxin site biosynthesis.blue type 1 copper ͉ purple copper CuA ͉ red type 2 copper ͉ folding ͉ kinetics

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Section: Discussionmentioning

confidence: 81%

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Savelieff¹,

Wilson²,

Youssef³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…[28][29][30] However, surprisingly, application of depsipeptides in deciphering protein function is limited primarily to the study of α-helical hydrogen bonding networks or hydrogen bonds to substrates in catalysis, since esters display different hydrogen bonding properties than their amide counterparts. [31][32][33] Depsipeptides are also known for their increased susceptibility to base hydrolysis compared to their peptide counterparts, and some studies have utilized this property within proteins. [34][35][36][37] Conversely, because of their lability most reported Fmoc-based solid phase peptide synthesis (SPPS) methods for depsipeptides typically involve extensive optimization to minimize hydrolysis and reduce racemization observed when the standard protocols are used for ester synthesis.…”

Section: Hhmi Author Manuscriptmentioning

confidence: 99%

Probing the role of the backbone carbonyl interaction with the Cu_A center in azurin by replacing the peptide bond with an ester linkage

et al. 2017

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The role of a backbone carbonyl interaction with an engineered Cu A center in azurin was investigated by developing a method of synthesis and incorporation of a depsipeptide where one of the amide bonds in azurin is replaced by an ester bond using expressed protein ligation. Studies by electronic absorption and electron paramagnetic resonance spectroscopic techniques indicate that, while the substitution does not significantly alter the geometry of the site, it weakens the axial interaction to the Cu A center and strengthens the Cu-Cu bond, as evidenced by the blue shift of the near-IR absorption that has been assigned to the Cu-Cu ψ→ψ* transition. Interestingly, the changes in the electronic structure from the replacement did not result in a change in the reduction potential of the Cu A center, suggesting that the diamond core structure of Cu 2 S Cys2 is resistant to variations in axial interactions.Exploring the structure and function of metalloproteins requires intimate knowledge about the roles of residues around the metal-binding sites. Such knowledge forms a strong basis for successful design and engineering of novel metalloproteins with tunable functional properties. [1][2][3][4][5][6] The most common way to acquire such knowledge is site directed mutagenesis (SDM). Because SDM is limited to only ∼20 proteinogenic amino acids, determining the precise role of the residues is difficult, as SDM often simultaneously changes more than one factor, such as electronic and steric effects, at the same time. Therefore, it is nearly impossible to de-convolute the contributions of each individual factor in such instances. To overcome this limitation, non-proteinogenic analogs of natural amino acids have been incorporated into metalloproteins. [7][8][9][10][11] For example, we have previously employed Expressed Protein Ligation (EPL) to replace conserved metal-binding amino acids with their isostructural nonproteinogenic analogs. 12,13 Such replacements allowed us to de-convolute steric factors from other factors such as electronic effects and hydrophobicity, thereby firmly establishing the roles of these residues in metalloprotein function. 12 HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptWhile most residues use side chains to interact with metal centers, increasing numbers of examples have appeared in the literature showing backbone carbonyl oxygens as metalcoordinating ligands. One such example is the Cu A center found in cytochrome c oxidase (CcO) and nitrous oxide reductase (N 2 OR). [17][18][19][20] Cu A contains a dinuclear copper center bridged by two cysteine thiolates wherein each copper is also coordinated by a histidine (Figure 1). More interestingly, perpendicular to this "diamond core" structural plane, are three carbonyl oxygens of the backbone peptide linkage that are within 2.17 to 4.07 Å of the two copper centers, suggesting that these backbone carbonyl oxygens may play a key role in the formation and fine-tuning of the Cu A center. 21 However, their roles in CcO, N 2 OR...

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“…The ligand exchange and metal-and/or cluster-type conversion might have been key evolutionary events from an archetypal mononuclear metalloprotein module toward a modern, high potential Rieske protein subunit of the respiratory complexes III, in which one of two histidyl ligands plays crucial electron/ proton transfer roles in the quinol-oxidizing Q o -site catalysis (16,18). Likewise, the dinuclear copper center in the Cu A site of mitochondrial and bacterial respiratory complexes IV, which is the primary electron acceptor site from mobile cytochrome c, is known to be structurally and evolutionarily related to the mononuclear copper proteins such as azurin and plastocyanin in the photosynthetic electron transfer chain (45)(46)(47)(48)(49)(50)(51). Possible common prototypal evolutionary patterns in both cases are (i) the mono-to dinuclear metal center conversion with a few residue substitutions in the immediate metal-binding site and (ii) the modular evolution from water-soluble (mononucleartype) ancestral protein modules, with minimal changes of the native protein scaffolds (Fig.…”

Section: Fig 2 Comparative Visible Near-uv Absorption Spectra Of Thmentioning

confidence: 99%

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

Iwasaki

Kounosu

et al. 2005

Journal of Biological Chemistry

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Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Riesketype ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe 3؉/2؉ center at the engineered local metal-binding site (Zn 2؉ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.Natural selection allows the utilization of a limited number of protein scaffolds to produce proteins with different types of active sites for various biological catalysis, molecular recognition, and metabolic needs. Metal ions add new functionality to proteins, facilitating some of the most difficult biological catalytic reactions (1-3). For this reason, design and engineering of a specific metalbinding site in natural and de novo protein scaffolds to promote new and specific functionalities are attractive targets in the field of basic and applied protein design research (4, 5).The metallosulfur redox sites, containing sulfurs, usually from cysteinyl side chains, are the most common in biological redox-active metalloproteins (1). In particular, the iron-sulfur (Fe-S) proteins are widely distributed over all living organisms and have been considered to be of early evolutionary origin (2, 6, 7). The mononuclear iron core is the simplest form of Fe-S redox sites, present in small modular proteins such as rubredoxin (Rd) 1 and desulforedoxin. These proteins have the iron atom coordinated in an approximately tetrahedral geometry to the sulfur atoms of four cysteinyl residues (1). Other major forms of protein-bound Fe-S redox sites are polynuclear clusters (such as [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters) for which some specific synthesis/assembly enzymes are required for in vivo cluster formation and maturation steps (7-10). These sites are also found within complex metalloprotein molecules themselves where they form part of the internal electron transfer conduit to or from the catalytic site (e.g. in some membrane-bound respiratory complexes (11-13)) as a result of modular evolution.Among the biological Fe-S clusters with at least one noncysteinyl ligand, "Rieske-type" [2Fe-2S] clusters are ubiquitous in a vari...

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Transformation of the Cu_A Redox Site in Cytochrome c Oxidase into a Mononuclear Copper Center

Cited by 37 publications

References 26 publications

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Probing the role of the backbone carbonyl interaction with the Cu_A center in azurin by replacing the peptide bond with an ester linkage

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

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Transformation of the CuA Redox Site in Cytochrome c Oxidase into a Mononuclear Copper Center

Cited by 37 publications

References 26 publications

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase

Probing the role of the backbone carbonyl interaction with the CuA center in azurin by replacing the peptide bond with an ester linkage

Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold

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Transformation of the Cu_A Redox Site in Cytochrome c Oxidase into a Mononuclear Copper Center

Probing the role of the backbone carbonyl interaction with the Cu_A center in azurin by replacing the peptide bond with an ester linkage