Luminescence of deoxyhemocyanin and deoxytyrosinase.

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 263 No. 20 Ieaue of July 15 p 95769577,1988 0 1988 by The American Society’for Bi&hemistryand MbPecular Biology, Inc. Printed in U.S.A.

Luminescence of Deoxyhemocyanin and Deoxytyrosinase” (Received for publication, March 21,1988)

Thomas N. SorrellS, Mariano Beltramini, and Konrad Lerch From the Biochemisches Znstitut der Uniuersitat Zurich, Zurich, Switzerland

10 nM. The tyrosinase samples were reduced with a 2-fold excess of hydrogen peroxide. Each solution was placed into a 50-ml bulb which was connected directly to a fluorescence cuvette and had a side arm containing a stopcock. The apparatus and solution were cooled in an ice bath anddeoxygenated by evacuating it atapproximately 10 Torr for 1-2 min with gentle swirling, then refilling the flask with argon. The procedure was repeated at least 5 times, and then the solution was transferred directly into thecell while under vacuum by inverting the apparatus. The carbonyl derivatives were prepared by admitting CO to the cuvette. Fluorescence spectra were recorded on a Spex Fluorolog spectrometer.

RESULTS AND DISCUSSION Thedeoxyformofhemocyaninsandtyrosinases from certain species displays a weak low-energy luPrevious work on copper(1) complexes having unsaturated minescence when solutions of the protein are irradiheterocyclic ligands has demonstrated that luminescence ocated with light at approximately 290 nm. Theemission curs by emission from a metal-to-ligand charge transfer state most likely results from a copper-to-imidazole charge (10, 16, 17). However, the absence of an emission signal may transfer state as shown by studies with a synthetic result from nonradiative quenching of the excited state. In copper(1) complexhaving three imidazole ligands. synthetic systems, this usually occurs as a resultof interaction between solute and solvent, and itcan be overcome by working at low temperature where the solutions are frozen as a glass. Spectroscopic and physicochemical studies of hemocyanin In proteins, the quenching may result from the presence of and tyrosinase indicate that the binuclear copper sites of water or a neighboring amino acid residue like tryptophan. those proteins are very similar (1, 2). One hemocyanin from Thus, failure to observe luminescence in reduced copper proPanulirus interruptus has been characterized by x-ray crys- teins may not be related to the ligation of the metal ion but tallography; and its active site, in the reduced deoxy form, rather may reflect the environment proximal to themetal ion. A synthetic copper(1) complex having three imidazole licontains two copper ions, each coordinated to three histidyl gands shows a luminescence at lower energy and reduced imidazole residues (3). Considering the homology of protein intensity compared to thatobserved for its carbonyl derivative sequences among many species, it is likely that one of the copper ions (so-called “CUB”)has the same substructure in all (10). The observation that three imidazole ligands are apparently bound to thecopper ions in at least one hemocyanin (3) hemocyanins and tyrosinases (4). The reduced forms of copper proteins aregenerally difficult suggested to us that a luminescence, similar to that for the to examine by the traditionally utilized spectroscopic methods synthetic species, should be observed for the protein. Excitation with light at 290 nm results in a weak luminescence for because the dlo copper(1) ionis colorless and diamagnetic. However, it was discovered that the carbonyl derivative of hemocyanin and tyrosinase from several species as summahemocyanin (5-8) and tyrosinase (9) luminesces at around rized in Table I. Representative spectra are shown in Figs. 1 550 nm when the proteins are illuminated with light having a and 2. The intensity of the emission for the deoxy proteins is wavelength near 300 nm. Wehave used thisproperty to between 30 and 100 times less than that observed for the evaluate a syntheticmodel having a copper(1) ion coordinated to three imidazole groups and CO (10). Since the synthetic carbonyl derivative. This may account for the failure to observe it previously ( 5 , 10). Addition of O2causes immediate compound also shows a luminescence in the absence of carbon monoxide, in apparent contrast to the proteins (5, IO), we quenching of the signal resulting from the charge transfer transition; and, as observed previously ( 5 ) , addition of either have re-examined the possibility that deoxyhemocyanin and O2 or CO results in loss of intensity for the tryptophan tyrosinase luminesce under conditions used to study the carbonyl derivative. In this paper we report that certain species luminescence centered at about 330 nm. show a weak luminescence at low energy which we attribute TABLE I to emission from a copper-to-imidazole charge transfer state.

Emission datafor copper proteinsand model h,

MATERIALS AND METHODS

Species”

The tyrosinases (Neurospora crassa (11) and Streptomyces glauces e m (12)) and hemocyanins (Octopus uulgaris (13), Carcinus muenas (14), and Limulus polyphemus (15)) were isolated and purified by literature procedures. The protein solutions were prepared in appropriate buffers to give a final protein concentration of approximately

Carbonvl nm

Octopus vulgaris‘ Limulus polyphemus’ Carcinus maenas’ 556 Streptomyces glaucesens‘ Neurospora crassa‘540 [Cu(timm)+l$ 550

* This work was supported by the Schweizerische Nationalfonds Grant 3.236-0.85 and theKanton of Zurich. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Fellow of the Alfred P.Sloan Foundation, 1985-1987. On leave from the Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290.

Deoxv

640 628

537 545 540

556 592

Room temperature in buffer. Hemocyanin. e Tyrosinase. In methanol-ethanol glass at 77 K (Ref. 10). timm is tris[2-(1methylimidazolyl)]methoxymethane.

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Luminescence of Deoxyhemocyanin /*

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450

600

750

Wavelength, nm

FIG. 1. Emission spectra for hemocyanin from C. maenas. deoxy; - - -, oxy; and -. -.-, carbonyl. The emission signal for the carbonyl derivativeis shown a t one-tenth of its actual intensity.

-,

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whereas the arthropodan hemocyanins are more similar to the fungal tyrosinase from Neurospora, all of which luminesce intheir deoxy forms. Presumably,some difference inthe structure of the protein of the molluscan hemocyanin influences the active site, resulting in quenching of luminescence. Whether the quenchingreflects a difference in ligation of the copper ions or in the protein environment is uncertain at present. The emission for the carbonyl derivative of proteins from different species occurs at nearly the same wavelength; however, luminescence maxima for the deoxy proteins vary considerably. Again, whether this difference reflects changes in the local environment of the active site or coordination by ligating groups other than histidine is uncertain. However, the observation of luminescence for the deoxy state provides another probe of the active site structure that can be used to evaluate putativemodels for these binuclear copper proteins. Acknowledgment-Acknowledgment is made to the University of North Carolina for a Pogue Research Leave (to T. N. S.). REFERENCES 1. Solomon, E. I. (1981) in Copper Proteins (Spiro, T. G., ed) pp.

-.

41-108, John Wiley & Sons, New York 2. Lerch, K. (1987) Life Chem. Reports 5 , 221-234 3. Gavkema. W. P. J.. Volbeda., A,., and Hol. G. J. W. (1986) . , J. Mol. Biol. 187, 255-275 4. Lerch. K.. Huber. M.. Schneider. H.-J.. Drexel. R.. and Limen. B. (1986) J . Inorg. kochem. 26, 213-217 5. Kuiper, H. A., Finazzi-Agro, A., Antonini, E., and Brunori, M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,2387-2389 6.Finazzi-Agro, A., Zolla, L., Flamigni, L., Kuiper, H. A., and Brunori, M. (1982) Biochemistry 21, 415-418 7. Zolla, L., Calabrese, L., and Brunori, M.(1984) Biochim. Biophys. Acta 788,206-213 8. Zolla, L., Kuiper, H. A., Finazzi-Agro, A., and Brunori, M. (1984) J . Inorg. Biochem. 22, 143-148 9. Kuiper, H.A., Lerch, K., Brunori, M., and Finazzi-Agro, A. (1980) FEBS Lett. 111,232-234 10. Sorrell, T. N., and Borovik, A. S. (1987) J . Am. Chern. SOC.109, 4255-4260 11. Lerch, K. (1987) Methods Enzymol. 142, 165-169 550 Li! Wavelength. nm 12. Lerch, K., and Ettlinger, L. (1972) Eur. J. Biochem. 31,427-437 13. Salvato, B., Ghiretti-Magaldi, A., and Ghiretti, F. (1979) BioFIG. 2. Emission spectra for tyrosinase from N. crassa. chemistry 18, 2731-2736 -, deoxy; - - -, oxy; and -. -. -, carbonyl. The emission signal for 14. Salvato, B., Ghiretti-Magaldi, A., and Ghiretti, F. (1974) Biothe carbonyl derivative shown is a t one-fiftieth of its actual intensity. chemistry 13, 4778-4783 15. Ghiretti-Magaldi, A,, Nuzzolo, C., and Ghiretti, F. (1966) Biochemistry 5 , 1943-1951 For both the molluscan hemocyanin from Octopus and the D. R., Kirchoff, J . R., and Goodwin, K. V. (1985) bacterial tyrosinase from Streptomyces, we observe no lumi- 16.McMillin, Coord. Chem. Reu. 64,83-92 nescence for the deoxy form. It is noteworthy that thosetwo 17. Sorrell, T . N., and Borovik, A. S. (1987) Inorg. Chem. 26, 1957proteinsare very similarintheir physical properties (4), 1964 /'

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