The spectroscopic and electrochemical properties of blue copper proteins are strikingly different from those of inorganic copper complexes in aqueous solution. Over three decades ago this unusual behavior was ascribed to constrained coordination in the folded protein; consistent with this view, crystal structure determinations of blue proteins have demonstrated that the ligand positions are essentially unchanged on reduction as well as in the apoprotein. Blue copper reduction potentials are tuned to match the particular function of a given protein by exclusion of water from the metal site and strict control of the positions of axial ligands in the folded structure. Extensive experimental work has established that the reorganization energy of a prototypal protein, Pseudomonas aeruginosa azurin, is approximately 0.7 eV, a value that is much lower than those of inorganic copper complexes in aqueous solution. The lowered reorganization energy in the protein, which is attributable to constrained coordination, is critically important for function, since the driving forces for electron transfer often are low (approximately 0.1 eV) between blue copper centers and distant (>10 A) donors and acceptors.

The oxidized blue copper proteins azurin and stellacyanin have been investigated through 1H NMR at 800 MHz and the results compared with those for plastocyanin (Bertini, I.; Ciurli, S.; Dikiy, A.; Gasanov, R.; Luchinat, C.; Martini, G.; Safarov, N. J. Am. Chem. Soc. 1999, 121, 2037). By exploiting saturation transfer between the oxidized and the reduced forms, all the hyperfine shifted signals can be assigned, including the β-CH2 protons of the coordinated cysteines, which are so broad not to be detected under direct observation. Both hyperfine shifts and line widths of the latter signals differ dramatically from one protein to another:  average hyperfine shifts of about 850, 600, and 400 ppm and average line widths of 1.2, 0.45, and 0.25 MHz are observed for azurin, plastocyanin, and stellacyanin, in that order. The observation of a nuclear line width of 1.2 MHz is unprecedented in high-resolution NMR in solution. These data are interpreted as a measure of the out-of-plane displacement of the copper ion, which increases on passing from azurin to plastocyanin to stellacyanin. The present approach seems general for the investigation of blue copper proteins.

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The unique spectroscopic properties of blue-copper centers, i.e. the strong charge-transfer band at approximately 600 nm and the narrow hyperfine coupling in the EPR spectrum, are reviewed. The concept of rack-induced bonding is summarized. The tertiary structure of the protein creates a preformed chelating site with very little flexibility, the geometry of which is in conflict with that preferred by Cuz+. The structure of the metal site in azurin is discussed. It is shown that the three strong ligands, one thiolate S and two imidazole N, are in a configuration intermediate between those preferred by Cu2+ and Cu'. It is emphasized that cysteine is an obligatory component of a blue site, whereas the weak interaction with a methionine S is not necessary. The minimum rack energy is estimated to be 70 kJ . mol-'. It is pointed out that the high reduction potentials of bluecopper centers are a result of the protein-forced ligand-field-destabilized site structure. It is suggested that the potentials are tuned by variations in n back bonding, and this is supported by a linear increase in ALF (ligand field) with decreasing electron-transfer enthalpy. Site-directed mutagenesis has shown that large hydrophobic residues in the site increase the potential, whereas negative groups or water decrease it. It is also shown that the fine-tuning of the properties of the metal site by rackinduced bonding can alter the electron-transfer reorganization energy. Kinetic results with azurin mutants support a through-bond tunneling mechanism for intramolecular electron transfer in proteins. Finally, it is pointed out that the concept of rack-induced bonding is a universal principle of macromolecular structure/function relationships, which should be applied also to other systems.

A spectroelectrochemical method has been used to determine the reduction potential of the copper site in wild-type and 22 mutant forms of azurin from Pseudomonas aeruginosa at 25 "C and in the range pH 4-8; the effect of buffers and ionic strength on the potentials has also been studied. Amino-acid residues changed include Metl21, which provides an S atom at a distance of about 0.3 nm from the metal, some amino acids in the hydrophobic patch, other residues believed to be important in electron transfer with physiological partners and some internal amino acids. The observed potentials span a range of about 300 mV. In all cases the potentials increase with decreasing pH, but the pK, values describing the pH dependence are essentially unchanged except in three mutants, where they change by pH 0.6-1.1 (up in one and down in two). The largest potential changes were found in some Met121 mutants, at which position large hydrophobic residues raise the potential, whereas negatively charged residues lower it; a decreased potential is also found in the Met121 + End mutant, which probably has H,O coordinated to the metal. Gly45 has its carbonyl group coordinated to copper, but the potential of Gly45 3 Ala is close to that of the wild type. Some substitutions in the hydrophobic patch cause an increase in the potential, whereas substitutions involving His35 and Glu91 do not result in significant changes. No single mechanism for tuning the potential of the copper site can be discerned, but in many cases there are probably indirect effects of the protein conformation causing changes in metal-ligand interactions.Azurin belongs to a family of small blue-copper proteins which function in electron-transfer chains in plants and bacteria (Adman, 1985). These proteins have unique spectral properties, such as intense absorbance bands around 600 nm and a narrow hypefine splitting in the CuZ+ EPR signal, and they also have unusually high reduction potentials among Cu2+ complexes. A perplexing fact is, however, that these potentials can vary by more than 0.5 V between members of the family despite the fact that the spectroscopic properties are relatively constant. Since the intense blue color and specific EPR signal are properties of the oxidized proteins, it has been suggested that proteins with a high reduction potential have a preferential stabilization of the Cu+ form (Gray and Malmstrom, 1983). To test this hypothesis, or other possible mechanisms for tuning the potentials, is one of the purposes of the program in site-directed mutagenesis of which the study described here is part.We have prepared mutant forms of azurin with substitutions of a metal ligand (Metl21) (Karlsson et al., 1991), of surface residues, some believed important in the interactions with electron-transfer partners , and also of some residues localized more internally. In this study we report the determination by thin-layer spectroelectrochemisCorrespondence to T. Vanngird,

Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of Cu I in the unfolded protein is linear or trigonal, whereas Cu II prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline) 2 2؉͞؉ reference system.The folding of a protein to its native three-dimensional structure is a spontaneous process, driven by the tendency of the peptide chain to assume the conformation of minimum free energy. As first clearly enunciated by Lumry and Eyring in 1954 (1, 2), the universal minimum for a given protein (i.e., for a specific amino acid sequence) may be reached at the expense of some local energy maximum. They further suggested that evolution has availed itself of this so-called rack phenomenon to create strain and distortion in prosthetic groups or coenzymes, thereby tuning the electronic properties by the mechanical force. This idea also led to a visualization of evolutionary fine tuning of active-site properties in protein superfamilies by small variations in amino acid sequences. The idea of conformationally induced strain in protein active sites was further developed both by Lumry himself (3) and by other authors. Vallee and Williams (4) stressed, in particular, how strain in the active site of the ground state of a catalytic metalloenzyme (e.g., a blue copper protein) can poise the metal ion for its reaction with substrate. The unique properties of blue copper were first described in 1960 (5), and they were attributed to a rack mechanism by one of us in 1964 (6). The first attempt to estimate the rack energy for blue copper, based on ligand-field considerations, was published in 1983 (7), and recently, Brill (8) has developed a model to calculate the mechanical energy associated with stress and strain and applied it to one specific blue protein, azurin. Interestingly, electronic structure calculations (9, 10) and spectroscopic experiments (9) have suggested...

Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

Resonance Raman spectra of the single-copper blue proteins azurin (from the bacteria Pseudomonas putida, Pseudomonas aeruginosa, Iwasaki sp., Bortadella pertussis, Bortadella bronchiseptica, and Alcaligenes faecalis), plastocyanin (from French bean and spinach), and stellacyanin (from the Japanese and Chinese lacquer trees Rhus vernicifera) and the multicopper oxidases lacease (from the fungus Polyporus versicolor and the Japanese and Chinese lacquer trees), ascorbate oxidase (from zucchini squash), and ceruloplasmin (from human blood serum) are reported. Cryoresonance Raman observations (10-77 K) are reported for selected azurins, stellacyanin, the plastocyanins, and the laceases. Isotope studies employing 63Cu/65Cu and H/D substitution are reported for selected azurins and stellacyanin, allowing identification of modes having significant