3859
Inorg. Chem. 1988, 27, 3859-3861
Notes Contribution from the Departments of Chemistry and Biochemistry and the Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, and Department of Chemistry, Amherst College, Amherst, Massachusetts 01002
Copper X-ray Absorption Spectroscopic Studies of the Bovine Plasma Amine Oxidase-Sulfide Complex
1
I
8950
8970
Robert A. Scott,' Cheryl E. Cot&,2and David M. Dooley*s2
8990 9010 Energy (eV)
9030
9050
Received May 19, 1988
Bovine plasma amine oxidase is a copper-containing enzyme ~ also contains an organic (2 Cu atoms/protein m o l e c ~ l e )which cofactor t h a t displays t h e reactivity of a carbonyl group but t h a t has proved difficult to definitively identify. There is now considerable evidence that pyrroloquinoline quinone (PQQ), or a closely similar derivative, is the organic cofactor in this class of amine oxidase^.^-^ Both t h e copper and t h e organic cofactor are required for activity, and recent work is consistent with a 1:l:l copper:cofactor:subunit stoichiometry.1° Substrate amines react with the organic cofactor and are oxidized to aldehydes, thereby reducing the enzyme by two electrons; copper is required for the subsequent oxidation of the reduced enzyme by 02, producing H 2 0 2and (generally) releasing NH3.3 Elucidating the structural and functional relationships between the cofactors is therefore critical for any fundamental understanding of amine oxidase catalysis.
NMR" and fluorescence energy-transfer datal2 indicate that the copper ions and the reactive quinone carbonyl group(s) are separated by several angstroms. Nevertheless, numerous experiments point to significant interactions between copper and the ~ 0 f a c t o r . l ~In particular, the irreversible inactivation of bovine
(1) University of Georgia (2) Amherst College. (3) For recent reviews on the molecular properties, mechanisms, and bio-
(4)
(5) (6) (7)
(8)
(9)
logical roles of copper-containing amine oxidases see: (a) Knowles, P. F.; Yadav, K. D. S. In Copper Proteins and Copper Enzymes; Lontie, R., Ed.; CRC: Boca Raton, FL, 1984; Vol. 2, pp 103-129. (b) Mondovi, B., Ed. Structure and Functions of Amine Oxidases; CRC: Boca Raton, FL, 1985. Lobenstein-Verbeek, C. L.; Jongejan, J. A.; Frank, J.; Duine, J. A. FEES Lett. 1984, 107, 305-309. Ameyama, M.; Hayashi, M.; Matsushita, K.; Shinagawa, E.; Adachi, 0. Agric. Biol. Chem. 1984, 48, 561-565. Moog, R. S.; McGuirl, M. A.; Cote, C. E.; Dooley, D. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8435-8439. (a) Williamson, P. R.; Moog, R. S.; Dooley, D. M.; Kagan, H. M. J . Biol.Chem. 1986, 261, 16302-16305. (b) Knowles, P. F.; Pandeya, K. B.; Rius, F. X.;Spencer, C. M.; Moog, R. S.; McGuirl, M. A.; Dooley, D. M. Biochem. J . 1987, 241, 603-608. (a) Van der Meer, R. A.; Jongejan, J. A.; Frank, J., Jr.; Duine, J. A. FEBS Lert. 1986, 206, 1 1 1-1 14. (b) Van der Meer, R. A,; Duine, J. A. Biochem. J . 1986,239,789-791. (c) Van der Meer, R. A.; Jongejan, J. A.; Duine, J. A. FEES Lett. 1987, 221, 299-304. Glatz, Z.; Kovar, J.; Macholh, L.; Pec, P. Biochem. J . 1987, 242,
603-607. (10) (a) Klinman, J. P.; Janes, S., unpublished observations. (b) Cote, C.
E.; McGuirl, M. A,; Knowles, P. F.; Singh, I.; Dooley, D. M., unpublished observations. (1 1) Williams, T. J.; Falk, M. C., Biochem. J . 1986, 261, 15949-15954. (12) Lamkin, M. S.; Williams, T. J.; Falk, M.C. Arch. Biochem. Biophys. 1988, 261, 72-79.
0020-1669/88/1327-3859$01.50/0
-8' 3.0
"
5.0
"
7.0 k
"
9.0
"
11.0
"
13.0
(A.')
Figure 1. Copper K absorption edge (a) and EXAFS (b) spectra for resting (oxidized) bovine plasma amine oxidase (-) and its complex with sulfide (- - -).
plasma amine oxidase by sulfide14and the effects of cyanide on the substrate-reduced amine oxidasesi5indicate that the electron transfer between t h e copper ions and PQQ is possible. In t h e former case, HS- was proposed to initially bind to the enzyme, forming a Cu"-SH- complex, which then underwent further reactions to eventually give Cu(1) and t h e reduced organic cofactor as products. Given the key role suggested for the CuII-SHcomplex in t h e inactivation reaction, we decided to characterize the structure more completely by using X-ray absorption spectroscopy (XAS). A great deal of work has established that both t h e copper K-edge and extended X-ray absorption fine structure (EXAFS) are quite sensitive to (and hence diagnostic for) sulfur c~ordination.'~J~ Experimental Section Bovine plasma amine oxidase was purified by modifications of published procedures.18 Samples used for XAS were highly homogeneous as judged by SDS and gradient polyacrylamide gel electrophoresis; specific activities exceeded 0.25 unit mg-I, assayed with benzylamine. (One enzyme unit catalyzes the oxidation of 2 rmol min-I of benzylamine to
(1 3) (a) Mondovi, B. In Advances in Inorganic Biochemistry; Eichhorn, G. L., Marzilli, L. G., Eds.; Elsevier: New York, 1984; Vol. 6, pp 225-244. (b) Suzuki, S.; Sakurai, T.; Nakahara, A,; Manabe, T.;Okuyama, T. Biochemistry 1983, 22, 1630-1635. (c) Mondovi, B.; Sabatini, S.; Befani, 0. J . Mol. Catal. 1984, 23, 325-330. (d) Rinaldi, A.; Floris,
G.; Sabatini, S.; Finazzi-Agrd, A,; Giartosio, A,; Rotilio, G.; Mondovi, B. Biochem. Biophys. Res. Commun. 1983,115,841-848. (e) Kluetz, M. D.; Adamsons, K.; Flynn, J. E., Jr. Biochemistry 1980, 19, 1617-1621. ( f ) Finazzi-AgrB,A.; Rindaldi, A.; Floris, G.; Rotilio, G. FEES Lett. 1984,176,378-380. (g) Suzuki, S.; Sakurai, T.; Nakahara, A.; Manabe, T.; Okuyama, T. Biochemistry 1986, 25, 338-341. (h) Mondovi, B. Biochem. J . 1987, Morpurgo, L.; Agostinelli, E.; Befani, 0.; 248, 865-870. (14) Dooley, D. M.; Cote, C. E. J . Biol. Chem. 1984, 259, 2923-2926. (15) Dooley, D. M.; McGuirl, M. A,; Peisach, J.; McCracken, J. FEES Lett. 1987,-214, 274-278. (16) Scott, R. A. Methods Enzymol. 1985, 117, 414-459. (17) Scott, R. A.; Schwartz, J. R.;Cramer. S. P . Biochemistry 1986, 25, 5546-5555. (18) (a) Turini, P.; Sabatini, S.; Befani, 0.;Chimenti, F.; Casanova, C.; Riccio, P. L.; Mondovi, B. Anal. Biochem. 1982, 125, 294-298. (b) Summers, M. C.; Markovic, R.; Klinman, J. P. Biochemistry 1979, 18, 1969-1 979.
0 1988 American Chemical Society
3860 Inorganic Chemistry, Vol. 27, No. 21, 1988
Notes
Table I. Curve-Fitting Results for the First Coordination Spheres of Resting and Sulfide-Treated Bovine Plasma Amine Oxidase" CU-( N ,0) cu-s sample resting resting
+ sulfide
fit 1 2
N (4Y (3)
R, A
3 4 5
(3) (2) (4)
2.02 2.02 2.04
1.98 1.98
ha2,A2 +0.0019 +0.0002
(1)
+0.0026
(1)
-0.0000 +0.0052
ha2,A2
Yb
2.30
+0.0245
0.041 0.048
2.32 2.30
+0.0037 +0.002s
R, A
N
(1)
0.038 0.034 0.094
" N is the number of scatterers per copper; R is the copper-scatterer distance; Aa2 is the relative mean square deviation in R, Au2 = u*(sample) u2(reference), where the reference is [Cu(im),12+ at 4 K for Cu-(N,O) and [Cu(mnt),12- at 4 K for Cu-S (im = imidazole; mnt = maleonitriledithiolate). All fits were over the range k = 4.0-12.0 A-f. is a goodness-of-fit statistic normalized to the overall magnitude of the k'[x(k)] data: f =
E[ k ' ( x , d ) (k3x)mar - (k3x)min
Numbers in parentheses were not varied during optimization. benzaldehyde.) Amine oxidase samples for XAS were exhaustively dialyzed against metal-free 0.1 M KP04 buffer (pH 7.0) containing 2 mM EDTA followed by dialysis against the metal-free buffer alone. Solutions were concentrated to approximately 1 mM protein in a vertical, semipermeable membrane device from Biomolecular Dynamics. Glycerol was then added (in order to facilitate glass formation upon freezing), giving a final concentration of 0.75 mM protein ( 1 .S mM Cu). Trace metal ions were removed from the buffer by passage over a Chelex column; the effectiveness of this technique was confirmed by atomic absorption spectroscopy. Sodium sulfide solutions were prepared immediately before use. The sulfide complex of resting bovine plasma amine oxidase was prepared by adding a concentrated sulfide solution, sufficient to give a sulfide concentration equal to 2O[Cu], to the enzyme in the Lucite XAS cuvette and rapidly (
