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1.Intrinsic fluorescences of the sulfur-transferring protein rhodanese (or thiosulfate : cyanide sulfurtransferase) and of artificially persulfurated bovine serum albumin were studied. Both are attributable mainly to tryptophan side-chains. Addition of cyanide or sulfite to these proteins causes a 20-250/, increase in fluorescence.2. Both proteins exhibit a Iow intensity absorption band a t about 330 nm which disappears on addition of cyanide or sulfite. This absorption can be restored in rhodanese, after the treatment with cyanide, by excess thiosulfate ; in the same time the fluorescence decreases to that of sulfur-containing enzyme.3. A change in solvent viscosity, obtained by addition of glycerol, reduces the quenching effect in both proteins. On the basis of these data a long-range energy-transfer mechanism between the donor (tryptophan) and an acceptor is proposed. The acceptor involved seems to be one or more persulfide groups (R-SSH) in both proteins.

The luminescence of oxidized or reduced azurin, an intensely blue copper protein, has been observed. The addition of ha+ or Hg2+ reduced the luminescence of the apoprotein to one third of its value while Ag+ reduced it to two thirds. This quenching is explained in terms of tryptophan distorsion produced by the different coordination of these metal ions. A singlet-singlet internal conversion is postulated as the mechanism for luminescence quenching in all azurin derivatives. Copper is able to bind both the silver-and mercury-treated protein, as shown by electron paramagnetic resonance experiments, without restoring the native blue color. Moreover addition of copper reduced the luminescence in the silver-treated protein to that of the native protein; if NaCl is now added, the blue color is restored. This NaCl effect is absent in the mercury-treated apoprotein.These results suggest that the blue color is related to the binding of copper to a t least one of the ligands involved in the mercury or silver complexing, most probably a sulfur atom. Azurin is a well-known bacterial protein [I-31involved in electron transfer reactions. It contains a single copper atom per 16000 molecular weight and is intensely blue [4]. Previous work from this laboratory has demonstrated that azurin shows an unusual fluorescence emission band, centered a t 308 nm, which is attributed to its single tryptophan [3]. The fluorescence yield of the protein is increased three times by removal of copper. This fact was interpreted as a "heavy metal" effect. On this basis the tryptophan was postulated to be very near to copper in the active site of azurin. The present investigation was undertaken in order to obtain additional data which could support this interpretation ofthe mechanism of fluorescence quenching caused by the presence of copper. MATERIALS AND METHODSAll chemicals used were reagent grade, purchased from Merck A.G. (Darmstadt, Germany). Azurin and apoazurin were obtained from Pseudomonas fluorescens as previously described [3]. Spectrophotometric analyses were performed with a Beckman Ratio Recording Spectrophotometer Model DK2A. Fluorescence spectra were obtained with a Turner model 210 Xpectro using excitation and emission band-width of 2.5 or IOnm. The samples were maintained a t 20 "C by a circulating water bath. Phospho- Electron paramagnetic resonance (EPR) spectra were recorded on a Varian 4502-14 spectrometer, equipped with 100-kHz field modulation within a Varian multipurpose cavity, and variable-temperature accessory. Frequencies were measured with a Hewlett-Packard X 532 B frequency meter ; g values [6] were calculated by the Fieldial and the measured frequency. Circular dichroism (CD) spectra were recorded a t 26 "C using a Cary60 spectropolarimeter equipped with a 6001 CD attachment. The measurements were made in I-cm lightpath cells; the ellipticity was expressed as A e = EL -EB. RESULTS Fluorescence Quenching by Hg2+ and Ag+It was previously demonstrated [3] that apoazurin can bind stoichiometric amounts of Hg2+. This bi...

(1) 45% of the total copper of green zucchini ascorbate oxidase is EPR-detectable. At least two species of copper are present, one with a small A parallel (Type 1) and one with a large A parallel (Type 2). Computer simulated spectra indicated 50% contribution by each type of copper. (2) Azide inhibited ascorbate oxidase activity by an uncompetitive mechanism. EPR and optical spectra performed on titration of ascorbate oxidase with azide indicated the formation of a copper-azide complex. The Type 2 copper appears to be the binding site of azide. The involvement of the EPR non-detectable copper as an anion binding site with high affinity toward azide can not be excluded.

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