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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,

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(36)Cl(-) was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of (36)Cl(-), saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10(-4) s(-1) at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on (36)Cl(-), which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br(-) and NO3 (-). The slowly exchanging Cl(-) did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.