Blue Copper Protein Amicyanin Research Articles

Geometrical Description of Protein Structural Motifs.

We present a geometrical method that can identify secondary structural motifs in proteins via angular correlations. The method uses crystal structure coordinates to calculate angular and radial signatures of each residue relative to an external reference point as the number of nearest-neighbor residues increases. We apply our approach to the blue copper protein amicyanin using the copper cofactor as the external reference point. We define a signature termed Δβ which describes the change in angular correlation as the span of nearest neighbor residues increases. We find that three turn regions of amicyanin harbor residues with Δβ near zero, while residues in other secondary structures have Δβ greater than zero: for β-strands, Δβ changes gradually residue by residue along the strand. Extension of our analysis to other blue copper proteins demonstrated that the noted structural trends are general. Importantly, our geometrical description of the folded protein accounts for all forces holding the structure together. Through this analysis, we identified some of the turns in amicyanin as symmetrical anchor points.

Molecular dynamics of amicyanin reveals a conserved dynamical core for blue copper proteins

Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some beta-strands. However, they strictly maintain the arrangement of the portions of the beta-barrel that are conserved in the folding architecture of the blue copper proteins. Paracoccus versutus amicyanin equilibrates more rapidly, shows lower atomic deviation values, and is less rigid with respect to Paracoccus denitrificans amicyanin. Principal component analysis reveals that the conformational subspaces corresponding to eigenvectors with the same index for each of the two molecules are not necessarily equivalent. Nevertheless, a core scaffold with constrained dynamics exist for both amicyanins. In addition, two fairly flexible regions that are located on the opposite side with respect to the interaction sites with the partner molecules in the redox process have been evidenced in the protein structure. This description of amicyanin, with a few mobile regions remote from the active site and a rigid scaffold including most of the protein beta-barrel, has a close similarity with that of azurin and plastocyanin, two other cupredoxins previously investigated in simulation. Furthermore, similarities in the distribution of the atomic fluctuations indicate that amicyanin, azurin, and plastocyanin possess common dynamical features, in spite of differences in their structure. On the basis of these findings, we suggest that topological constraints imposed by the folding in correspondence of protein regions that are the most conserved determine the protein dynamics of the cupredoxin family. The dynamical properties of the cupredoxins might be controlled for functional advantages that include the binding mechanism with the biological partners and the collective inner motions of the protein matrix required for the electron transfer, whereas long-range conformational changes in the redox reaction should be excluded.

A preliminary time-of-flight neutron diffraction study on amicyanin fromParacoccus denitrificans

Crystals of the blue copper protein amicyanin suitable for neutron diffraction were grown by the sitting-drop method, followed by repeated macroseeding using solutions prepared with D(2)O. Although the crystal sizes were the same, crystals grown using solutions made up in H(2)O in the initial stages of macroseeding and solutions with D(2)O in later stages did not diffract neutrons well. However, when the protein was initially exchanged with buffered D(2)O and then crystallized and also macroseeded using solutions made up in D(2)O throughout, the crystals diffracted neutrons to high resolution. One of those crystals was used to collect a data set to a resolution of 1.9 A.

Interpretation of the temperature-dependent color of blue copper protein mutants

The electronic absorption spectrum of the mutant of the blue copper protein amicyanin with a pseudoazurin loop (AmiPse) shows a remarkable temperature dependence. The absorption band at ≈460 nm increases at low temperature while the transition at ≈600 nm is not much affected by a variation of the temperature. An approximate density functional theory (DFT) study of the active site model [CuII(imidazole)2(SCH3)(S(CH3)2)]+ (protein backbone and solvation neglected) leads to two local minimum structures (axial and rhomb) which both have a geometry close to that typical for blue copper proteins. One (rhomb) has two structurally different histidine donors, and this geometry is also found in most experimental type 1 structures. The two forms axial and rhomb are distortional isomers and are energetically almost degenerate. The temperature dependence of the spectrum of AmiPse is interpreted with a temperature-dependent change of the relative population of the two local minimum structures with slightly different energy. The 460 nm transition is believed to be due to preferential population of the structure rhomb; this is in agreement with the published assignment of the high energy transition, based on thorough spectroscopic and computational studies. Consequences of a perturbation of the “gas phase” structures axial and rhomb by the protein and solvation are also discussed on the basis of published, experimentally observed structures and spectroscopic data.

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan–tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H AB, related to electronic coupling between the two centers, and λ, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter β, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.

The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry.

The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa(red)=6.3 and pKa(ox) < or = 3.2 at 22 degrees C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s-1 at 25 degrees C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 degrees C, at which the rate constant for this conversion was determined to be 35 +/- 20 s-1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.

Loop-Directed Mutagenesis of the Blue Copper Protein Amicyanin fromParacoccus versutusand Its Effect on the Structure and the Activity of the Type-1 Copper Site

Four loop-mutants of the blue copper protein amicyanin from Paracoccus versutus have been constructed and characterized. The mutations replaced the loop containing three (Cys93, His96, Met99) of the four copper ligands in amicyanin by the “ligand loops” of P. aeruginosa azurin (AmiAzu), A. faecalis pseudoazurin (AmiPaz), P. nigra plastocyanin (AmiPcy), and P. aureofaciens nitrite reductase (AmiNiR). The copper centers of all variants appear to be perfect type-1 Cu sites although the AmiNiR variant exhibits diminished stability. The optical spectra of the AmiAzu and AmiPaz variants display a significant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of the three stable variants are all higher than that of wt amicyanin. The reduced forms of the loop-mutants protonate at the C-terminal histidine, with pKa values of 5.6 (AmiAzu), 5.4 (AmiPaz), and 5.7 (AmiPcy) (6.8 for wt amicyanin). AmiAzu is the first known cupredoxin with more than two amino acids between the cysteine and histidine ligands that undergoes this protonation. The electron self-exchange rate constants at 25 °C are 5.7−7.5 times lower for the loop mutants than for wt amicyanin (1.2 × 105 M-1 s-1). The results are interpreted by taking into consideration that three metal ligands are intimately connected with the stable β-sandwich structure of the cupredoxin. This leaves considerable freedom in positioning the fourth one, the C-terminal His, on the “ligand loop”. This explains the ease by which the C-terminal histidine ligand can be excised and replaced by external ligands without losing the metal binding property of the protein. The results also help understand the remarkable evolutionary success of the combination of cupredoxin fold and Cu site for mediating biological electron transfer.

Active-site mutants of the blue copper protein amicyanin from Thiobacillus versutus

Active-site mutants of the blue copper protein amicyanin from Thiobacillus versutus

Introduction of a Cu A site into the blue copper protein amicyanin from Thiobacillus versutus

The C-terminal loop of the blue copper protein amicyanin, which contains three of the four active site ligands, has been replaced with a Cu A binding loop. The purple protein produced has visible and EPR spectra identical to those of a Cu A centre. Recent evidence strongly suggests that the Cu A centre of cytochrome c oxidase and the A centre of nitrous oxide reductase are similar and are both binuclear. It therefore follows that the purple amicyanin mutant created here also possesses a binuclear Cu A centre.

Type 1 blue copper protein amicyanin from Thiobacillus versutus: line-broadening effects of chromium(III) complexes and related studies

The effect of redox-inactive cationic and anionic paramagnetic chromium(III) complexes on the 1H NMR spectrum of the reduced type 1 blue copper protein amicyanin, AmCuI, from Thiobacillus versutus has been studied as a means of defining sites for association at the protein surface. With [Cr(CN)6]3– two sites are detected, one at the adjacent hydrophobic patch close to the exposed imidazole of the co-ordinated His-96, and the other at Phe-92 which has Lys-59, Lys-60, Arg-69 and Arg-100 in close proximity and is adjacent to the active site-co-ordinated Cys-93. In contrast, the cationic complexes [Cr(NH3)6]3+ and [Cr(en)3]3+(en = ethane-1,2-diamine) cause no significant line broadening and no preferred sites for association are detected. Kinetic stopped-flow studies on the competitive inhibition by [Cr(CN)6]3– of the [Fe(CN)6]3– oxidation of AmCuI indicate that [Fe(CN)6]3– reacts at two sites, one of which is inhibited by [Cr(CN)6]3– and the other is unaffected by [Cr(CN)6]3–. It is suggested that the first of these corresponds to reaction at the Phe-92 site, contributing 25% to the reaction, and the second to reaction at His-96. Therefore, in its reaction with [Fe(CN)6]3– amicyanin has adjacent and remote binding sites.

Solution Structure of the Type 1 Blue Copper Protein Amicyanin from Thiobacillus versutus

A three-dimensional solution structure of amicyanin from Thiobacillus versutus has been determined by distance geometry and restrained molecular dynamics. A total of 984 experimentally derived constraints were used for the final refinement (881 distance constraints and 103 dihedral angle constraints). Stereospecific assignments were made for 17 prochiral β-methylene protons (33%) and the methyl groups of eight valine residues. Fourteen structures were selected to represent the solution structure. They show an average pairwise backbone root-mean-square deviation of 1·19 Å. The overall structure can be described as a β-sandwich, built up of nine β-strands. The copper atom is located between three loops on one end of the molecule. Two of these loops contribute the copper ligands. His54 is on the loop between β-strands 4 and 5. The other three ligands, Cys93, His96 and Met99, are located evenly spaced on the loop between β-strands 8 and 9. This loop is folded in two consecutive type 1 turns with His96 as the donor and acceptor of the NH i-CO i- 3 hydrogen bonds. The folding is reminiscent of the general cupredoxin fold. Considerably different are the large 21 residue N-terminal extension, that is unique to amicyanin and forms an extra β-strand (strand 1), and the region between β-strands 5 and 7. The partly surface-exposed copper ligand His96 is surrounded by a hydrophobic patch consisting of seven residues.

Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i.

The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

Crystal structure analysis and refinement at 2·15Å resolution of amicyanin, a type I blue copper protein, from Thiobacillus versutus

The crystal structure of the type I blue copper protein amicyanin from Thiobacillus versutus has been determined by Patterson search techniques on the basis of the molecular model of amicyanin from Paracoccus denitrificans, and refined by energy-restrained least-squares methods. Amicyanin crystallizes in the trigonal space group P3 2 with unit cell dimensions of a=b=87·40 A ̊ , c=38·20 A ̊ . The asymmetric unit is composed of three independent molecules centred on the crystallographic 3 2 axes. The final R-value is 17·4% for 15,984 reflections to a resolution of 2·15 Å. The polypeptide fold in amicyanin is based on the β-sandwich structure commonly found in blue copper proteins. Nine β strands are folded into two twisted β-sheets that pack together with a filling of non-polar residues between them. The geometry of the copper site is similar to that of plastocyanin. There are four ligands, arranged approximately as a distorted tetrahedron, to the copper atom: His54, Cys93, His96 and Met99. One of the copper ligands, His96, is exposed to the surface and lies in the centre of a cluster of seven hydrophobic residues.

Redox reactivity of the type 1 (blue) copper protein amicyanin from Thiobacillus versutus with inorganic complexes

Electron-transfer reactions of the type 1 copper protein amicyanin from Thiobacillus versutus with [Co(terpy)2]2+(terpy = 2, 2′:6′, 2″-terpyridine) as a reductant for AmCuII, and [Fe(CN)6]3– and [Co(phen)3]3+(phen = 1, 10-phenanthroline) as oxidants for AmCuI have been explored by stopped-flow kinetic studies at 25 °C, I= 0.100 M (NaCl). The reaction with [Co(terpy)2]2+ is a straightforward single-stage process, whereas both the oxidaltions give biphasic kinetics. The first stages of the latter exhibit an active-site ‘switch-off’ mechanism previously assigned as a protonation (and dissociation) of the His-96 ligand of AmCuI. From the studies with [Co(phen)3]3+ an acid dissociation constant pKa of 6.6 and rate constant at the higher pH of 7.1 × 103 M –1 s–1 are obtained. The reaction with [Fe(CN)6]3–gives similar behaviour with a pKa, of 6.6, and from the fitting procedure adopted, a rate constant at the higher pH of 8.3 × 1 06 M–1s–1, which is at the upper limit of the stopped-flow range. The second stage of the reaction is explained by the formation of a less-reactive form of AmCuI in amounts of between 5 and 40% depending on the pH. The rate law in the case of [Co(phen)3]3+ is independent of oxidant concentration, shows little dependence on pH and gives rate constants (a) in the range 0.07–0.12 s–1. For the more reactive [Fe(CN)6]3– the rate equation is k2obs=a+b[Fe(CN)63–], with a varying between 0.1 3 and 0.1 5 s–1 and b= 680 M–1s–1. The similar values of a for the two reactions are consistent with a common rate-controlling intramolecular isomerisation step. The bimolecular rate constant b is ≈ 104 times smaller than the rate constant for the first stage of the [Fe(CN)6]3– oxidaltion at pH ≈ 8. The nature of the structural differences between the two forms of AmCuI is considered.

Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.

Cloning, sequencing and expression studies of the genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase from Thiobacillus versutus

The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyanin-encoding gene is in agreement with the published protein sequence. The gene codes for a pre-protein with a 25-amino-acid-long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified beta-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.

The structural homology of amicyanin from Thiobacillus versutus to plant plastocyanins.

The complete amino acid sequence of the blue copper protein amicyanin of Thiobacillus versutus, induced when the bacterium is grown on methylamine, has been determined as follows: QDKITVTSEKPVAAADVPADAVVVGIEKMKYLTPEVTIKAGETVYWVNGEVMPHNVA FKKGIVGEDAFRGEMMTKDQAYAITFNEAGSYDYFCTPHPFMRGKVIVE. The four copper ligand residues in this 106-residue-containing polypeptide chain are His54, Cys93, His96, and Met99. The Thiobacillus amicyanin is 52% similar to the amicyanin of Pseudomonas AM1, the only other copper protein known with the same spacing between the second histidine ligand and the methionine ligand. T. versutus amicyanin contains no cysteine bridge and is more closely related to the plant copper protein plastocyanin than to the bacterial copper protein azurin. Alignment of the two known amicyanin sequences with the consensus sequence of the plastocyanins and comparison with the known three-dimensional structure of poplar leaves plastocyanin reveals that the bacterial proteins have the same overall structure with two beta-sheets packed face to face. The major structural differences between the amicyanins and the plastocyanins appear to be located in two of the five loops that connect the six identified beta-strands of the amicyanins. The first of these two loops, connecting strands F and G, contains a ligand histidine and must have a different conformation from the same loop in the plastocyanins because it is shorter by two amino acids. Further differences occur in the loop connecting the strands D and E. This loop contains only 17 residues in amicyanin whereas the corresponding loop of plastocyanin contains 25 residues. Despite these differences the amicyanins appear much closer related to the plastocyanins than to the azurins. The present findings demonstrate that the occurrence of blue copper proteins with clearly plastocyanin-like features is not restricted to photosynthetic redox chains.

PH-dependent redox activity and fluxionality of the copper site in amicyanin from Thiobacillus yersutus as studied by 300- and 600- MHz 1H NMR.

The kinetics of the deuteronation of one of the copper ligand histidines of the reduced Type I blue-copper protein amicyanin from Thiobacillus versutus was studied as a function of temperature by 300- and 600- MHz 1H NMR. The NMR data were analyzed with the help of a three site exchange model. Deuteron exchange between the histidine ligand and the solution appears to be catalyzed by phosphate. After deuteronation the histidine can occur in two conformations. The electron self-exchange rate of amicyanin was determined as a function of temperature and ionic strength. At 298 K, pD = 8.6, I = 0.05 M, the ese rate amounts to 1.3 x 10(5) M-1 S-1. The activation parameters amount to delta H not equal to = (52 +/- 3) kJ/mol and delta S not equal to = (26 +/- 9) J/mol.K. The dependence of the ese rate on ionic strength is small. The deuteronated amicyanin appears to be redox-inactive. The experimental findings clearly distinguish amicyanin from other classes of blue-copper proteins like the azurins and the pseudo-azurins.

A 1H-NMR study on the blue copper protein amicyanin from Thiobacillus versutus. Resonance identifications, structural rearrangements and determination of the electron self-exchange rate constant.

A number of resonances in the 1H-NMR spectra of reduced and oxidised amicyanin from Thiobacillus versutus have been identified by one- and two-dimensional NMR techniques. The second-order electron self-exchange rate constant (8.5 x 10(4) M-1.s-1; pH = 7.4; T = 308.5 K) was determined by measuring the line broadening of six singlets in slightly oxidised solutions of the protein. A large increase in electron exchange rate is observed in the presence of ferrocyanide. The copper atom in the reactive centre of the protein appears to be coordinated by nitrogens from two histidines and sulfurs from a methionine and a cysteine. One of the ligand histidines becomes protonated at low pH [pK*a = 6.74 (+/- 0.02)], the asterisk indicating value uncorrected for the deuterium isotope effect] in reduced amicyanin. This is the first example of a non-photosynthetic blue copper protein in which a ligand histidine becomes protonated at low pH. A small pH-independent conformational rearrangement occurs upon oxidation.