Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.     

Raman study of reduced nicotinamide adenine dinucleotide bound to liver alcohol dehydrogenase

We report the first Raman spectra of reduced nicotinamide adenine dinucleotide (NADH) when bound to an enzymatic active site, that of liver alcohol dehydrogenase (LADH). This was obtained by subtracting the Raman spectrum of LADH from that of the binary LADH/NADH complex. There are significant changes in the spectrum of bound NADH as compared to that in solution. The data indicate that both the nicotinamide moiety and the adenine moiety are involved in the binding. At least one of the two NH2 moieties of NADH also participates.     

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.     

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.     

Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species.

Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.     

Spectroscopic characterization of native and Co(II)-substituted zucchini mavicyanin

Mavicyanin from zucchini peelings has been characterized by electronic absorption, circular dichroism (CD), magnetic circular dichroism (MCD), resonance Raman (RR), and electron paramagnetic resonance (EPR) spectra. The electronic absorption, CD, MCD, and EPR spectra are appreciably similar to those of stellacyanin from lacquer, in which the tetrahedral Cu center has a donor set composed of four amino acid residues [2 histidine (His), cysteine (Cys), and glutamine (Gln)]. Under neutral conditions, mavicyanin and stellacyanin show intense blue bands at 599 and 604?nm, respectively. However, the RR spectrum of mavicyanin between 300 and 450?cm^–1, which is believed to originate from the predominant Cu–S stretching vibration, is remarkably different from that of stellacyanin. This might be due to a slight distortion of the tetrahedral Cu(II) center toward tetragonal geometry in mavicyanin. Moreover, the d–d transition bands of Co(II)-substituted mavicyanin are slightly blue-shifted compared with those of Co(II)-substituted stellacyanin. This finding also suggests a difference in distortion between these tetrahedral Co(II) centers in spite of the same donor sets.     

The role of blue copper proteins in the oxidation of methylamine by an obligate methylotroph.

Organism 4025, an obligate methylotroph, when grown on methylamine in the presence of a high concentration of copper, contained high concentrations of methylamine dehydrogenase and two blue copper proteins, amicyanin and an azurin-type protein; these were purified to homogeneity and characterized. The methylamine dehydrogenase is a basic protein (pI 8.8) and consists of light and heavy subunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differed slightly from other methylamine dehydrogenases in its absorption spectrum and in its lack of thermal stability. Amicyanin, the more abundant blue copper protein, had an Mr of 11500, a midpoint redox potential of 294mV at pH 7.0, and a much lower isoelectric point (pI5.3) than other amicyanins. Its absorption maximum was 620 nm (7-24 nm higher than those of other amicyanins); its absorption coefficient (at 620 nm) was 3.8 mM-1 X cm-1. The 'azurin' (6% of the blue copper protein) had an Mr of 12500, a midpoint redox potential of 323 mV and a high isoelectric point (pI 9.4). Its absorption maximum was 620 nm, the absorption coefficient (16 mM-1 X cm-1) at this wavelength being considerably greater than that of any blue copper protein described previously. The partially-purified soluble cytochromes cH and cL were similar to those of other methylotrophs. The interactions of the purified redox proteins were investigated in order to elucidate their role in methylamine oxidation. Methylamine dehydrogenase was able to donate electrons only to amicyanin, the rate of reaction being 2.04 mmol/min per mumol of methylamine dehydrogenase; this is sufficient to account for the rate of respiration in whole bacteria. The blue copper proteins were able to react rapidly with each other and with both the soluble cytochromes c.     

The relationship of some copper (II) complexes of facultative tetrathioethers to the coordination environment in the "blue" copper proteins

The facultative potentially tetradentate thioether ligands 1,2-bis(methylthioethylthio)ethane (2,2,2), 1,3-bis(2-methylthioethylthio)propane (2,3,2) and 1,2-bis(3-methylthiopropylthio)ethane (3,2,3) react with copper(II) salts to form Cu2(2,2,2)Cl4, Cu3(ligand)X6 (ligand = 2,3,2 and 3,2,3 X = Cl; ligand = 2,2,2 2,3,2 and 3,2,3 X = Br), and Cu(ligand)(ClO4)2. The stoichiometry and structures of these complexes are discussed in terms of the steric demands of the ligand and the nature of the halide. The [Cu(2,3,2)(ClO4)] ClO4 and [Cu(3,2,3)(ClO4) [ClO4 complexes have electronic spectra which exhibit the intense 600 nm band characteristic of the "blue" copper proteins. In fact, the spectrum of [Cu(2,3,2)(ClO4)]ClO4 is very similar to that of pseudomonas aeroginosa azurin.     

Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase

Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Tuning the geometries of a de novo blue copper protein by axial interactions

The axial interactions of Cu(2+) in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu(2+), exhibiting a blue or green color. The UV-vis spectral bands were observed at approximately 600?nm and approximately 450?nm, with the A (~450)/A (~600) ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu-S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.     

Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.     

Isolation and characterization of a blue copper protein from Thiobacillus versutus

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1.     

DNA interaction of new copper(II) complexes with sulfonamides as ligands

New copper(II) complexes with sulfonamide ligands have been prepared and characterized. Sulfonamide ligands were prepared through a reaction between 8-aminoquinoline and either 2-mesitylene (Hqmesa), 4-tert-butylbenzene (Hqtbsa), or alpha-toluene (Halphaqtsa) sulfonyl chlorides. The structural analysis carried out for complex [Cu(alphaqtsa)(2)] indicated that the local environment of the Cu(II) cation is between a square planar and a tetrahedral geometry, with stacking of the benzene rings of the sulfonyl ligands between neighbor molecules. Powder EPR spectra at room temperature gave rhombic spectra for the [Cu(alphaqtsa)(2)] and [Cu(qmesa)(2)] complexes and an axial spectrum for the [Cu(qtbsa)(2)] complex, probably due to the steric hindrance of the methyl groups. Complexes [Cu(alphaqtsa)(2)] and [Cu(qmesa)(2)] are artificial chemical nucleases that degrade DNA in the presence of sodium ascorbate. A study of the radical scavengers revealed that the ROS (reactive oxygen species) involved in the DNA damage were hydroxyl, singlet oxygen-like species, and superoxide anion.     

Oxidation of (horse) hemoglobin by copper: an intermediate detected by electron spin resonance.

The oxidation of horse hemoglobin by Cu(II) has been followed by the changes in the electron spin resonance spectra of copper. By stopped-flow and freeze-quenching techniques, it is shown that the second-order rate constant for the binding of Cu(II) to hemoglobin is greater than 5 X 10(5) mol-1 s-1 and the apparent first-order rate for the reduction of Cu(II) to Cu(I) is 0.051 s-1. It is also shown that the binding of Cu(II) to hemoglobin is followed by an alteration of the Cu(II) spectrum, decreasing the g values. This process has an apparent rate constant of 17 s-1 and presumably involves a conformational change in the region of the copper binding site. It is also shown that this conformational change is apparently necessary for Cu(II) to oxidize hemoglobin.     

Resonance Raman spectroscopic studies of adriamycin and copper(II)-adriamycin and copper(II)-adriamycin-DNA complexes

Characteristic resonance Raman spectra are observed on ionization of the phenolic groups in adriamycin. On the basis of these results, vibrational assignments for the Raman bands of adriamycin are reported. Distinct Raman spectra are observed for Cu(II)-adriamycin complexes at pH approximately 5 and pH approximately 13. The data indicate that at lower pH a bis complex of Cu(II) is formed, which transforms to a polymeric Cu(II) chelate at higher pH. Upon interaction of the metal-drug complex with calf thymus DNA at pH approximately 5, a ternary complex is formed in which the Cu(II)-complexed adriamycin is intercalated into DNA.     

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic 1H NMR study of Co(II)-pseudoazurin

Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family. The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase. The copper at the active site is coordinated to His40, Cys78, His81 and Met86 in a distorted tetragonal geometry. We have recorded and assigned the (1)H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes. The (1)H NMR spectrum of Co(II)-pseudoazurin closely resembles that of Co(II)-rusticyanin, reflecting an altered conformation for the Met-Co(II)-Cys moiety in both proteins, compared to Co(II)-azurin, amicyanin and stellacyanin. The electron spin density onto the Sgamma(Cys) is larger in Co(II)-pseudoazurin compared to Co(II)-rusticyanin. Instead, the Co(II)-Met interaction is similar in both derivatives. Hence, the different metal-ligand interactions might be independently modulated by the protein structure. The present work also shows that the electron spin density onto the Co(II)-S(cys) bond is sensibly smaller than the Cu(II)-S(cys). Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins.     

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins)

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.