Spectroscopic studies of the Met182Thr mutant of nitrite reductase: role of the axial ligand in the geometric and electronic structure of blue and green copper sites

A combination of spectroscopic methods and density functional calculations has been used to describe the electronic structure of the axial mutant (Met182Thr) of Rhodobacter sphaeroides nitrite reductase in which the axial methionine has been changed to a threonine. This mutation results in a dramatic change in the geometric and electronic structure of the copper site. The electronic absorption data imply that the type 1 site in the mutant is like a typical blue copper site in contrast to the wild-type site, which is green. Similar ligand field strength in the mutant and the wild type (from MCD spectra) explains the similar EPR parameters for very different electronic structures. Resonance Raman shows that the Cu-S(Cys) bond is stronger in the mutant relative to the wild type. From a combination of absorption, CD, MCD, and EPR data, the loss of the strong axial thioether (present in the wild-type site) results in an increase of the equatorial thiolate-Cu interaction and the site becomes less tetragonal. Spectroscopically calibrated density functional calculations were used to provide additional insight into the role of the axial ligand. The calculations reproduce well the experimental ground-state bonding and the changes in going from a green to a blue site along this coupled distortion coordinate. Geometry optimizations at the weak and strong axial ligand limits show that the bonding of the axial thioether is the key factor in determining the structure of the ground state. A comparison of plastocyanin (blue), wild-type nitrite reductase (green), and the Met182Thr mutant (blue) sites enables evaluation of the role of the axial ligand in the geometric and electronic structure of type 1 copper sites, which can affect the electron-transfer properties of these sites.     

Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.     

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.     

Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site

A variety of spectroscopic techniques, combined with density functional calculations, are used to describe the electronic structure of the Leu513His variant of the type 1 Cu site in Myceliophthora thermophila laccase. This mutation changes the type 1 Cu from a blue to a green site. Electron paramagnetic resonance (EPR), optical absorption, circular dichroism, and magnetic circular dichroism (MCD) spectroscopies reveal that, relative to the trigonal planar blue type 1 Cu site in wild-type fungal laccase, the covalency and the ligand field strength at the Leu513His green type 1 Cu center decrease. Additionally, there is a significant reorientation of the d(x)()()2(-)(y)()()2( )singly occupied MO, such that the overlap with the Cys sulfur valence orbital changes from pi to sigma. A density functional study in which internal coordinates are systematically altered reveals that these changes are due to the increased strength of the axial ligand (none to His), leading to a tetragonal distortion and elongation of the equatorial Cu-ligand bonds. These calculations provide insight into the experimental differences in the EPR parameters, charge-transfer absorption spectrum, and ligand-field MCD spectrum between the axial-His variant and blue Cu centers (plastocyanin and the type 1 site in fungal laccase). There are also significant differences between the green site in the Leu513His variant and other naturally occurring, green type 1 Cu sites such as in nitrite reductase, which have short axial Cu-S(Met) bonds. The large difference in EPR parameters between these green type 1 sites derives from a change in ligand field excitation energies observed by MCD, which reflects a decrease in ligand field strength. This is associated with different steric interactions of a His vs an axial Met ligand in a tetragonally distorted type 1 site. Changes in the electronic structure of the Cu site correlate with the difference in reactivity of the green His variant relative to blue wild-type fungal laccase.     

Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond intradiol dioxygenases

The geometric and electronic structure of NO bound to reduced protocatechuate 3,4-dioxygenase and its substrate (3,4-dihydroxybenzoate, PCA) complex have been examined by X-ray absorption (XAS), UV-vis absorption (Abs), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The results are compared to those previously published on model complexes described as [FeNO]7 systems in which an S = 5/2 ferric center is antiferromagnetically coupled to an S = 1 NO-. XAS pre-edge analysis indicates that the Fe-NO units in FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] lack the greatly increased pre-edge intensity representative of most [FeNO]7 model sites. Furthermore, from extended X-ray absorption fine structure (EXAFS) analysis, the FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] active sites are shown to have an Fe-NO distance of at least 1.91 A, approximately 0.2 A greater than those found in the model complexes. The weakened Fe-NO bond is consistent with the overall lengthening of the bond lengths and the fact that VTVH MCD data show that NO(-)-->FeIII CT transitions are no longer polarized along the z-axis of the zero-field splitting tensor. The weaker Fe-NO bond derives from the strong donor interaction of the endogenous phenolate and substrate catecholate ligands, which is observed from the increased intensity in the CT region relative to that of [FeNO]7 model complexes, and from the shift in XAS edge position to lower energy. As NO is an analogue of O2, the effect of endogenous ligand donor strength on the Fe-NO bond has important implications with respect to O2 activation by non-heme iron enzymes.     

Determinants of the relative reduction potentials of type-1 copper sites in proteins

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.     

Ligand and loop variations at type 1 copper sites: influence on structure and reactivity

Type 1 (T1) copper sites promote biological electron transfer and are found in the cupredoxins and a number of copper-containing enzymes including the multi-copper oxidases. A T1 copper site usually has a distorted tetrahedral geometry with strong ligands provided by the thiolate sulfur of a Cys and the imidazole nitrogens of two His residues. The active site structure is typically completed by a weak axial Met ligand (a second weak axial interaction is found in azurin resulting in a trigonal bipyramidal geometry). The axial Met is not conserved and Gln, Phe, Leu and Val are also found in this position. Three of the four ligands at a T1 copper site are situated on a single C-terminal loop whose length and structure varies. Studies are discussed which investigate both the influence of physiologically relevant axial ligand alterations, and also of mutations to the length and structure of the ligand-containing loop, on the properties of T1 copper sites.     

Crystal structures of oxidized and reduced stellacyanin from horseradish roots

Umecyanin (UMC) is a type 1 copper-containing protein which originates from horseradish roots and belongs to the stellacyanin subclass of the phytocyanins, a ubiquitous family of plant cupredoxins. The crystal structures of Cu(II) and Cu(I) UMC have been determined at 1.9 and 1.8 A, respectively. The protein has an overall fold similar to those of other phytocyanins. At the active site the cupric ion is coordinated by the N(delta1) atoms of His44 and His90, the S(gamma) of Cys85, and the O(epsilon)(1) of Gln95 in a distorted tetrahedral geometry. Both His ligands are solvent exposed and are surrounded by nonpolar and polar side chains on the protein surface. Thus, UMC does not possess a distinct hydrophobic patch close to the active site in contrast to almost all other cupredoxins. UMC has a large surface acidic patch situated approximately 10-30 A from the active site. The structure of Cu(I) UMC is the first determined for a reduced phytocyanin and demonstrates that the coordination environment of the cuprous ion is more trigonal pyramidal. This subtle change in geometry is primarily due to the Cu-N(delta1)(His44) and Cu-O(epsilon1)(Gln95) bond lengths increasing from 2.0 and 2.3 A in Cu(II) UMC to 2.2 and 2.5 A, respectively, in the reduced form, as a consequence of slight rotations of the His44 and Gln95 side chains. The limited structural changes upon redox interconversion at the active site of this stellacyanin are analogous to those observed in a typical type 1 copper site with an axial Met ligand and along with its surface features suggest a role for UMC in interprotein electron transfer.     

The active-site structure of umecyanin, the stellacyanin from horseradish roots

The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a weakly interacting axial Met, giving a distorted tetrahedral geometry. Natural variations to this coordination environment are known, and we have utilized paramagnetic (1)H NMR spectroscopy to study the active-site structure of umecyanin (UMC), a stellacyanin with an axial Gln ligand. The assigned spectra of the Cu(II) UMC and its Ni(II) derivative [Ni(II) UMC] demonstrate that this protein has the typical His(2)Cys equatorial coordination observed in other structurally characterized cupredoxins. The NMR spectrum of the Cu(II) protein does not exhibit any paramagnetically shifted resonances from the axial ligand, showing that this residue does not contribute to the singly occupied molecular orbital (SOMO) in Cu(II) UMC. The assigned paramagnetic (1)H NMR spectrum of Ni(II) UMC demonstrates that the axial Gln ligand coordinates in a monodentate fashion via its side-chain amide oxygen atom. The alkaline transition, a feature common to stellacyanins, influences all of the ligating residues but does not alter the coordination mode of the axial Gln ligand in UMC. The structural features which result in Cu(II) UMC possessing a classic type 1 site as compared to the perturbed type 1 center observed for other stellacyanins do not have a significant influence on the paramagnetic (1)H NMR spectra of the Cu(II) or Ni(II) proteins.     

Active Site Models for the Cu(A) Site of Peptidylglycine α-Hydroxylating Monooxygenase and Dopamine β-Monooxygenase

A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated product. The present results have indicated that a superoxo species having a four-coordinate distorted tetrahedral geometry could be reactive enough to induce the direct C-H bond activation of aliphatic substrates in the enzymatic systems.     

X-ray absorption edge spectroscopy and computational studies on LCuO2 species: Superoxide-Cu(II) versus peroxide-Cu(III) bonding

The geometric and electronic structures of two mononuclear CuO2 complexes, [Cu(O2){HB(3-Ad-5-(i)Prpz)3}] (1) and [Cu(O2)(beta-diketiminate)] (2), have been evaluated using Cu K- and L-edge X-ray absorption spectroscopy (XAS) studies in combination with valence bond configuration interaction (VBCI) simulations and spin-unrestricted broken symmetry density functional theory (DFT) calculations. Cu K- and L-edge XAS data indicate the Cu(II) and Cu(III) nature of 1 and 2, respectively. The total integrated intensity under the L-edges shows that the 's in 1 and 2 contain 20% and 28% Cu character, respectively, indicative of very covalent ground states in both complexes, although more so in 1. Two-state VBCI simulations also indicate that the ground state in 2 has more Cu (/3d8) character. DFT calculations show that the in both complexes is dominated by O2(n-) character, although the O2(n-) character is higher in 1. It is shown that the ligand L plays an important role in modulating Cu-O2 bonding in these LCuO2 systems and tunes the ground states of 1 and 2 to have dominant Cu(II)-superoxide-like and Cu(III)-peroxide-like character, respectively. The contributions of ligand field (LF) and the charge on the absorbing atom in the molecule (Q(mol)M) to L- and K-edge energy shifts are evaluated using DFT and time-dependent DFT calculations. It is found that LF makes a dominant contribution to the edge energy shift, while the effect of Q(mol)M is minor. The charge on the Cu in the Cu(III) complex is found to be similar to that in Cu(II) complexes, which indicates a much stronger interaction with the ligand, leading to extensive charge transfer.     

CS2在Cu-S键中的插入反应的研究3:[Cu(S2CSC6H2Bu3^t-2,4,6]4.4C2H5OH的合成与晶体结构

2,4,6-三特丁基硫酚铜[Cu(SC6H2Bu3^t-2,4,6-)]n与CS2的反应产物, 用CS2-乙醇重结晶得黄色晶体, 经X射线单晶衍射法测定, 其结构为[Cu(S2CSC6H2Bu3^t-2,4,6-)].晶体属四方晶系, 空间群142d.a=b=1.7079(3)nm,c=3.5228(10)nm, V=10.275(6)nm3, Dc=1.19gcm^-3, Z=4.1863个衍射点参与修正,R=0.0559.四个铜原子形成变形四面体, CS2在全部四个Cu-S键中插入, 形成硫代黄原酸配体(RSCS2^-).配体中两个硫原子参与配位, 其中一个S桥连两个Cu原子, 另一个S端位于第三个Cu原子。每个Cu原子都采取平面三配位方式。CS2插入形成的CS2基团基本平面, C-S平均键长为0.1712nm。本文还对CS2, 在Cu-S和Ag-S键中插入反应产物的结构特点及影响反应的因素进行了讨论。     

Axial methionine has much less influence on reduction potentials in a CuA center than in a blue copper center

The role of the highly conserved axial methionine of the purple CuA center in an engineered CuA azurin on modulating the reduction potentials of the copper center was investigated by a systematic replacement of the methionine with glutamate, aspartate, and leucine. In contrast to the same substitutions in the structurally related blue copper azurin, much smaller changes in reduction potential were observed in the CuA azurin upon replacing the methionine ligand with negatively charged Glu (-8 mV) and Asp (-5 mV) and more hydrophobic Leu (+16 mV). These findings are important in understanding the different roles of the two cupredoxins. The diamond core Cu2S2(Cys) structure of the CuA is much more resistant to variations of axial ligand interactions than the distorted tetrahedral structure of the blue copper protein. This difference may translate into a much wider range of reduction potentials (>1000 mV) for blue copper proteins that transfer electrons to a variety of partners in many different biological systems and a much narrower range of reduction potentials (<40 mV) for CuA proteins where a small difference in reduction potentials between the CuA and its redox partners is required.     

Creation of a type 1 blue copper site within a de novo coiled-coil protein scaffold

Type 1 blue copper proteins uniquely coordinate Cu(2+) in a trigonal planar geometry, formed by three strong equatorial ligands, His, His, and Cys, in the protein. We designed a stable Cu(2+) coordination scaffold composed of a four-stranded α-helical coiled-coil structure. Two His residues and one Cys residue were situated to form the trigonal planar geometry and to coordinate the Cu(2+) in the hydrophobic core of the scaffold. The protein bound Cu(2+), displayed a blue color, and exhibited UV-vis spectra with a maximum of 602-616 nm, arising from the thiolate-Cu(2+) ligand to metal charge transfer, depending on the exogenous axial ligand, Cl(-) or HPO(4)(2-). The protein-Cu(2+) complex also showed unresolved small A(∥) values in the electron paramagnetic resonance (EPR) spectral analysis and a 328 mV (vs normal hydrogen electrode, NHE) redox potential with a fast electron reaction rate. The X-ray absorption spectrum revealed that the Cu(2+) coordination environment was identical to that found in natural type 1 blue copper proteins. The extended X-ray absorption fine structure (EXAFS) analysis of the protein showed two typical Cu-N(His) at around 1.9-2.0 ?, Cu-S(Cys) at 2.3 ?, and a long Cu-Cl at a 2.66 ?, which are also characteristic of the natural type 1 blue copper proteins.     

Spectroscopic and density functional theory studies of the blue-copper site in M121SeM and C112SeC azurin: Cu-Se versus Cu-S bonding

S K-edge X-ray absorption, UV-vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII-thioether model complex shows that the 38% S character in the ground state wave function of the blue-copper (BC) sites solely reflects the Cu-SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu-SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV-vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin-orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 A. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.     

Pyridazine based scorpionate ligand in a copper boratrane compound

Reaction of potassium tris(mercapto-tert-butylpyridazinyl)borate K[Tn(tBu)] with copper(II) chloride in dichloromethane at room temperature led to the diamagnetic copper boratrane compound [Cu{B(Pn(tBu))(3)}Cl] (Pn = pyridazine-3-thionyl) (1) under activation of the B-H bond and formation of a Cu-B dative bond. In contrast to this, stirring of the same ligand with copper(I) chloride in tetrahydrofuran (THF) gave the dimeric compound [Cu{Tn(tBu)}](2) (2) where one copper atom is coordinated by two sulfur atoms and one hydrogen atom of one ligand and one sulfur of the other ligand. Hereby, no activation of the B-H bond occurred but a 3-center-2-electron B-H···Cu bond is formed. The reaction of copper(II) chloride with K[Tn(tBu)] in water gave the same product 2, but a formal reduction of the metal center from Cu(II) to Cu(I) occurred. When adding tricyclohexyl phosphine to the reaction mixture of K[Tn(R)] (R = tBu, Me) and copper(I) chloride in MeOH, the distorted tetrahedral Cu complexes [Cu{Tn(R)}(PCy(3))] (R = tBu 3, Me 4) were formed. Compound 4 is exhibiting an "inverted" κ(3)-H,S,S, coordination mode. The copper boratrane 1 was further investigated by density functional theory (DFT) calculations for a better understanding of the M→B interaction involving the d(8) electron configuration of Cu.     

Design and spectroscopic characterization of peptide models for the plastocyanin copper-binding loop

The Cu(II)- and Co(II)-binding properties of two peptides, designed on the basis of the active site sequence and structure of the blue copper protein plastocyanin, are explored. Peptide BCP-A, Ac-Trp-(Gly)(3)-Ser-Tyr-Cys-Ser-Pro-His-Gln-Gly-Ala-Gly-Met-(Gly )(3)-His-(Gly)(2)-Lys-CONH(2), conserves the Cu-binding loop of plastocyanin containing three of the four copper ligands and has a flexible (Gly)(3) linker to the second His ligand. Peptide BCP-B, Ac-Trp-(Gly)(3)-Cys-Gly-His-Gly-Val-Pro-Ser-His-Gly-Met-Gly-CONH(2), contains all four blue copper ligands, with two on either side of a beta-turn. Both peptides form 1:1 complexes with Cu(II) through His and Cys ligands. BCP-A, the ligand loop, binds to Cu(II) in a tetrahedrally distorted square plane with axial solvent ligation, while BCP-B-Cu(II) has no tetrahedral distortion in aqueous solution. In methanolic solution, distortion of the square plane is evident for both BCP-Cu(II) complexes. Tetrahedral Co(II) complexes are observed for both peptides in aqueous solution but with 4:2 peptide:Co(II) stoichiometries as estimated by ultracentrifugation. Cu(II) reduction potentials for the aqueous peptide-Cu(II) complexes were measured to be +75 +/- 30 mV vs NHE for BCP-A-Cu(II) and -10 +/- 20 mV vs NHE for BCP-B-Cu(II). The results indicate that the plastocyanin ligand loop can act as a metal-binding site with His and Cys ligands in the absence of the remainder of the folded protein but, by itself, cannot stabilize a type 1 copper site, emphasizing the role of the protein matrix in protecting the Cu binding site from solvent exposure and the Cys from oxidation.     

Outer-sphere contributions to the electronic structure of type zero copper proteins

Bioinorganic canon states that active-site thiolate coordination promotes rapid electron transfer (ET) to and from type 1 copper proteins. In recent work, we have found that copper ET sites in proteins also can be constructed without thiolate ligation (called "type zero" sites). Here we report multifrequency electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopic data together with density functional theory (DFT) and spectroscopy-oriented configuration interaction (SORCI) calculations for type zero Pseudomonas aeruginosa azurin variants. Wild-type (type 1) and type zero copper centers experience virtually identical ligand fields. Moreover, O-donor covalency is enhanced in type zero centers relative that in the C112D (type 2) protein. At the same time, N-donor covalency is reduced in a similar fashion to type 1 centers. QM/MM and SORCI calculations show that the electronic structures of type zero and type 2 are intimately linked to the orientation and coordination mode of the carboxylate ligand, which in turn is influenced by outer-sphere hydrogen bonding.