Probing copper ligands in denatured <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> azurin: unfolding His117Gly and His46Gly mutants

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.     

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.     

The role of hydrogen bonding at the active site of a cupredoxin: the Phe114Pro azurin variant

The Phe114Pro mutation to the cupredoxin azurin (AZ) leads to a number of structural changes at the active site attributed to deletion of one of the hydrogen bonds to the Cys112 ligand, removal of the bulky phenyl group from the hydrophobic patch of the protein, and steric interactions made by the introduced Pro. The remaining hydrogen bond between the coordinating thiolate and the backbone amide of Asn47 is strengthened. At the type-1 copper site, the Cu(II)-O(Gly45) axial interaction decreases, while the metal moves out of the plane formed by the equatorial His46, Cys112, and His117 ligands, shortening the bond to the axially coordinating Met121. The resulting distorted tetrahedral geometry is distinct from the trigonal bipyramidal arrangement in the wild-type (WT) protein. The unique position of the main S(Cys) --> Cu(II) ligand-to-metal charge-transfer transition in AZ (628 nm) has shifted in the Phe114Pro variant to a value that is more typical for cupredoxins (599 nm). This probably occurs because of the removal of the Phe114-Cys112 hydrogen bond. The Phe114Pro mutation results in a 90 mV decrease in the reduction potential of AZ, and removal of the second hydrogen bond to the Cys ligand seems to be the major cause of this change. The C-terminal His117 ligand does not protonate in the reduced Phe114Pro AZ variant, which suggests that none of the structural features altered by the mutation are responsible for the absence of this effect in the WT protein. Upon reduction, the copper displaces further from the equatorial ligand plane and the Cu-S(Met121) bond length decreases. These changes are larger than those seen in the WT protein and contribute to the order of magnitude decrease in the intrinsic electron-transfer capabilities of the Phe114Pro variant.     

Snapshots of a dynamic folding nucleus in zinc-substituted Pseudomonas aeruginosa azurin

Zinc-substituted Pseudomonas aeruginosa azurin folds in two-state equilibrium and kinetic reactions. In the unfolded state, the zinc ion remains bound to the unfolded polypeptide via two native-state ligands (His117 and Cys112). The significantly curved Chevron plot for zinc-substituted azurin was earlier ascribed to movement of the folding-transition state. At low concentrations of denaturant, the transition state occurs early in the folding reaction (low Tanford beta-value), whereas at high-denaturant concentration, it moves closer to the native structure (high Tanford beta-value). Here, we use this movement to track the formation and growth of zinc-substituted azurin's folding nucleus with atomic resolution using protein engineering. The average phi (phi) value for 17 positions (covering all secondary-structure elements) goes from 0.25 in 0 M GuHCl (beta approximately 0.46) to 0.76 in 4 M GuHCl (beta approximately 0.86); a phi-value of 1 or 0 indicates native-like or unfolded-like interactions, respectively. Analysis of individual phi-values reveals a delocalized nucleus where structure condenses around a leading density centered on Leu50 in the core. The diffuse moving transition state for zinc-substituted azurin is in sharp contrast to the fixed polarized folding nucleus observed for apo-azurin. The dramatic difference in apparent kinetic behavior for the two forms of azurin can be rationalized as a minor alteration on a common free-energy profile that exhibits a broad activation barrier.     

Probing the reactivity of different forms of azurin by flavin photoreduction

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J.     

A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments

The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5?Å from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (τ_s) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. τ_s values of ≤1?ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5–15?ns for the tetragonal type 2 sites.     

Role of ligand substitution on long-range electron transfer in azurins.

Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin-imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways.     

Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.

Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.     

Active site modeling in copper azurin molecular dynamics simulations

Active site modeling in molecular dynamics simulations is investigated for the reduced state of copper azurin. Five simulation runs (5 ns each) were performed at room temperature to study the consequences of a mixed electrostatic/constrained modeling for the coordination between the metal and the polypeptide chain, using for the ligand residues a set of charges that is modified with respect to the apo form of the protein by the presence of the copper ion.The results show that the different charge values do not lead to relevant effects on the geometry of the active site of the protein, as long as bond distance constraints are used for all the five ligand atoms. The distance constraint on the O atom of Gly45 can be removed without altering the active site geometry. The coordination between Cu and the other axial ligand Met121 is outlined as being flexible. Differences are found between the bonds of the copper ion with the two apparently equivalent N¹ atoms of His46 and His117.The overall findings are discussed in connection with the issue of determining a model for the active site of azurin suitable to be used in molecular dynamics simulations under unfolding conditions. Figure Model of azurin active site. Copper ligand residues are cut off at C position except Gly45, for which the portion of backbone connecting it to His46 is shown. Only polar H atoms are shown. All atoms are in standard colors (Cu in violet), and the five ligands are labeled     

Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (相似文献   

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a beta-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D(2)O/H(2)O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide protons. In contrast, the last loop (including a short alpha-helix) and the last beta-strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.     

A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement

The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

 In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm^–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on ³⁴S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm^–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm^–1 are consistent with square-planar coordination. Addition of exogenous ¹⁵N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with ¹⁵N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with ¹⁵N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone. Received: 29 July 1996 / Accepted: 9 November 1996     

Ligand replacement study at the His120 site of purple CuA azurin

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.     

Structure of azurin from Alcaligenes denitrificans refinement at 1.8 A resolution and comparison of the two crystallographically independent molecules

The structure of the blue copper protein azurin, from Alcaligenes denitrificans, has been refined crystallographically by restrained least-squares methods. The final crystallographic R value for 21,980 observed reflections to 1.8 A (1 A = 0.1 nm) resolution is 0.157. The asymmetric unit of the crystal contains two independent azurin molecules, the model for which comprises 1973 protein atoms, together with three SO2-4 ions, and 281 water molecules. Comparison of the two molecules shows very high correspondence. For 125 out of 129 residues (excluding only the chain termini, residues 1 to 2 and 128 to 129) the root-mean-square (r.m.s.) deviation in main-chain atom positions is 0.27 A. For other structural parameters r.m.s. deviations are also low; torsion angles 6.5 degrees, hydrogen bond lengths 0.12 A, bonds to copper 0.04 A and bond angles at the copper 3.9 degrees. The only significant differences are at the chain termini and in several loops. Some of these can be attributed to crystal packing effects, others to genuine structural microheterogeneity. Refinement has confirmed that the copper co-ordination is best described as distorted trigonal planar, with strong in-plane bonds to His46 N delta 1, His117 N delta 1 and Cys112 S gamma, and much weaker axial interactions with Met121 S delta and Gly45 C = O. Two N-H...S hydrogen bonds characterize Cys112 S gamma as a thiolate (S-) sulphur and may influence the visible absorption maximum. Atoms in and around the copper site have very low mobility, whereas the most mobile regions of the molecule are the chain termini and some of the connecting loops between secondary structure elements, especially those at the "southern" end, remote from the copper site. Main-chain to side-chain hydrogen bonds supply important stabilizing interactions at the "northern" end. Surface features include the hydrophobic patch around His117, probably important for electron transfer, the SO2-4 site at His83, and the general absence of ion pairs, despite the presence of many charged amino acid residues. The 281 water molecules include 182 that occur as approximately twofold-related pairs. There are no internal water molecules. The water sites common to both azurin molecules include those in surface pockets and some in intermolecular contact regions. They are characterized by relatively low thermal parameters and numerous protein contacts.     

A spin-labeled derivative of Procion brilliant blue MX-R. Synthesis and interaction with pig heart nucleoside diphosphate kinase

The ¹H-NMR spectrum of cucumber basic blue protein (CBP) has been recorded. Examination of the spectrum of the reduced protein suggests that one or more sidechains exist in conformations which interconvert slowly at ambient temperatures. His 39, His 84 and Met 89 are identified as copper ligands by redox titration and by amino acid sequence homology with plastocyanin and azurin. The importance of a Phe sidechain close to the Met ligand in the potential blue copper site is confirmed. Broadening of His ligand resonances at elevated temperatures reveals an exchange process at the reduced copper centre.     

Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.     

Role of the axial ligand in type 1 Cu centers studied by point mutations of met148 in rusticyanin.

Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.