J. Mol. Biol. (1977) 112, 649-650
LETTERS TO THE EDITOR
Preliminary Crystallographic Data for a Basic Copper-containing Protein from Cucumber Seedlings A basic protein with a molecular weight of approximately 10,I00 and having a single atom of Type I copper has been isolated from cucumber seedlings. The oxidized, cupric, form of the protein has been crystallized as thin olive-green plates with space group P212121 and cell dimensions a ---- 30'8 A, b = 45.6/~ and o = 66.6 A. There is one protein molecule per asymmetric unit. There has been considerable interest both from biochemical and physicochemieal viewpoints for a number of years in the nature of the metal binding site in the so-called TyloeI copper proteins (see Fee, 1975 for a recent review). In the oxidized, cupric,form these proteins are characterized by a strong blue to blue.green colour arising from unusually intense visible absorption bands and by rather unique electron paramagnetic resonance properties having especially narrow hyperfine splittings. V~nfle several theories on the structure and function of this type of copper co-ordination site have been proposed, there has not yet appeared a detailed X-ray analysis of one of these proteins. We recently reported preliminary crystallographic data for poplar plastocyanin (Chapman c~ aI., 1977), a blue protein involved in photosynthetic electron transport, and wish to describe here preliminary crystallographic data on a new copper-containing protein from cucumber plants. The spectroscopic properties of the cupric form of this protein are also characteristic of Type I copper proteins but differ significantly in detail reflecting differences in the exact nature of the copper binding site. The cucumber protein was originally isolated and purified (Vickery, 1971) as a by-product of the extraction of indole-3-ethanol oxidase from young, light-grown cucumber seedlings (Vickery & Purves, 1972). The biological function of this protein is not known, but its properties (L. E. Vickery, manuscript in preparation) are closely similar to what may be identical proteins discovered in cucumber peelings (Markossian ctaL, 1974) and spinach leaves (Aikazyan & Nalbandyan, 1975), indicating that it may be widely distributed in green plant tissues. In contrast to plastocyanin, the new protein is basic, having a strong net positive charge near neutral pH. The molecular weight was determined to be 10,100±200 by gel filtration, sodium dodeeyl suIphate/polyacrylamide gel electrophoresis, sedimentation equilibrium and the amino acid composition. (A value of 8000 determined by gel filtration was reported by Markossian ct al., 1974.) The molecule consists of a single polypoptide chain of approximately 93 amino acids and contains one g-atom copper per mole. The absorption spectrum of the oxidized form has bands at 282 (280), 404 shoulder, 443 (442), 597 (592) and 750 nm t. The major visible band at 597 nm has a molar absorptivity of approximately 3500 (900) M-lcm -1. Unlike plastocyanin from the same species (Markossian et al., 1974), it does not exhibit the sharp fine structure in the aromatic Where two numerical values are given, the first is taken from data on our preparation, and the value in parenthesis is taken from Markossiane~ aL (1974). 649
650
P . M . COLMAN E T A L .
absorption region, and the ratio of A28o/A597 for the purified protein is 5.8 (12.8) due to the presence of t r y p t o p h a n in addition to tyrosine. Concentrated solutions appear green due to intense band(s) in the blue near 440 nm~ The electron parsmagnetic resonance spectrum of the Cu(II) form shows a strong rhombic distortion with gx----2"02, g~ ---- 2.08, g z = 2.207 and Ax----0"006, A~ ----0.001 and A z--0.0055 cm - I (g~l --~ 2.232, gm ---- 2.114, All = 0.005 cm-1); this is in contrast to the axial-type electron paramagnetic resonance spectrum of plastocyanin, b u t does resemble t h a t of stellacyanin (Peisach et al., 1967). The oxidized form of the protein shows no tendency to auto-reduce, and the reduced form is stable to air oxidation. Crystals suitable for X - r a y structure analysis were obtained from a concentrated solution of the protein (8 to 10 mg/ml) in 1.6 M-ammonium sulphate and 0.1 Msodium phosphate at p H 6-0. The protein solution was equilibrated with buffered a m m o n i u m sulphate solution at 4°C b y the v a p o r diffusion technique. The ammoninm sulphate concentration was raised from 1.6 M to 2"25 to 2"4 M during three to four weeks. The crystals which were formed under these conditions are thin olivegreen plates. S y m m e t r y and systematic absences on precession photographs show the space group to be P212121 with cell dimensions a = 30.8 A, b ---- 45.6 A, c = 66"6 A and V ---- 93,400 A a. The diffraction patterns extend to a resolution of at least 3 A. The crystals, like those of plastocyanin (Chapman et al., 1977), remain stable in the X - r a y beam for more t h a n one week. The partial specific volume inferred from the amino acid composition is 0.73 ml g - i ; the cell therefore contains four protein molecules which occupy 52% of the cell volume. The work was supported by grant 74/15398 from the Australian Research Grants Committee (to H. C. F.). One of the authors (P.M.C.) is a Queen Elizabeth I I Fellow. Department of Inorganic Chemistry University of Sydney Sydney 2006, Australia
PETER M. COLMAN H A N S C. F R E E M A N J. MITCHELL GUSS 1V/dTSUOMURATA VAX,ERIE A. NORRIS
JOHN A. M. RAMSHAW i~I.P. VEI~ATA~PPA~"
Department of Chemistry
LARRY E. VICKERY
University of California
Berkeley, Calif. 94720, U.S.A. Received 1 February 1977 REFERENCES Aikazyan, V. Ts. & Nalbandyan, R. M. (1975). P E B S Letters, 55, 272-274. Chapman, G. P., Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M., Norris, V. A., Ramshaw, J. A. M. & Venkatappa, M. P. (1977}. J. 111ol. Biol. 110, 187-189. Fee, J. A. (1975). Struct. Bond. 23, 1-60. Markossian, K. A., Aikazyan, V. Ts. & Nalbandyan, R. M. (1974). Biochim. Biophys. Acta, 359, 47-54. Peisach, J., Levine, W. G. & Blumberg, W. E. (19671. J. Biol. Chem. 242, 2847-2858. Vickery, L. E. (1971). Ph.D. Thesis, University of California, Santa Barbara. Vickery, L. E. & Purves, W. K. (1972). Plant Physiol. 49, 716-721. t Permanent address: Department of Chemistry, Karnatak University, Dharwar, India.
LETTERS TO THE EDITOR
Preliminary Crystallographic Data for a Basic Copper-containing Protein from Cucumber Seedlings A basic protein with a molecular weight of approximately 10,I00 and having a single atom of Type I copper has been isolated from cucumber seedlings. The oxidized, cupric, form of the protein has been crystallized as thin olive-green plates with space group P212121 and cell dimensions a ---- 30'8 A, b = 45.6/~ and o = 66.6 A. There is one protein molecule per asymmetric unit. There has been considerable interest both from biochemical and physicochemieal viewpoints for a number of years in the nature of the metal binding site in the so-called TyloeI copper proteins (see Fee, 1975 for a recent review). In the oxidized, cupric,form these proteins are characterized by a strong blue to blue.green colour arising from unusually intense visible absorption bands and by rather unique electron paramagnetic resonance properties having especially narrow hyperfine splittings. V~nfle several theories on the structure and function of this type of copper co-ordination site have been proposed, there has not yet appeared a detailed X-ray analysis of one of these proteins. We recently reported preliminary crystallographic data for poplar plastocyanin (Chapman c~ aI., 1977), a blue protein involved in photosynthetic electron transport, and wish to describe here preliminary crystallographic data on a new copper-containing protein from cucumber plants. The spectroscopic properties of the cupric form of this protein are also characteristic of Type I copper proteins but differ significantly in detail reflecting differences in the exact nature of the copper binding site. The cucumber protein was originally isolated and purified (Vickery, 1971) as a by-product of the extraction of indole-3-ethanol oxidase from young, light-grown cucumber seedlings (Vickery & Purves, 1972). The biological function of this protein is not known, but its properties (L. E. Vickery, manuscript in preparation) are closely similar to what may be identical proteins discovered in cucumber peelings (Markossian ctaL, 1974) and spinach leaves (Aikazyan & Nalbandyan, 1975), indicating that it may be widely distributed in green plant tissues. In contrast to plastocyanin, the new protein is basic, having a strong net positive charge near neutral pH. The molecular weight was determined to be 10,100±200 by gel filtration, sodium dodeeyl suIphate/polyacrylamide gel electrophoresis, sedimentation equilibrium and the amino acid composition. (A value of 8000 determined by gel filtration was reported by Markossian ct al., 1974.) The molecule consists of a single polypoptide chain of approximately 93 amino acids and contains one g-atom copper per mole. The absorption spectrum of the oxidized form has bands at 282 (280), 404 shoulder, 443 (442), 597 (592) and 750 nm t. The major visible band at 597 nm has a molar absorptivity of approximately 3500 (900) M-lcm -1. Unlike plastocyanin from the same species (Markossian et al., 1974), it does not exhibit the sharp fine structure in the aromatic Where two numerical values are given, the first is taken from data on our preparation, and the value in parenthesis is taken from Markossiane~ aL (1974). 649
650
P . M . COLMAN E T A L .
absorption region, and the ratio of A28o/A597 for the purified protein is 5.8 (12.8) due to the presence of t r y p t o p h a n in addition to tyrosine. Concentrated solutions appear green due to intense band(s) in the blue near 440 nm~ The electron parsmagnetic resonance spectrum of the Cu(II) form shows a strong rhombic distortion with gx----2"02, g~ ---- 2.08, g z = 2.207 and Ax----0"006, A~ ----0.001 and A z--0.0055 cm - I (g~l --~ 2.232, gm ---- 2.114, All = 0.005 cm-1); this is in contrast to the axial-type electron paramagnetic resonance spectrum of plastocyanin, b u t does resemble t h a t of stellacyanin (Peisach et al., 1967). The oxidized form of the protein shows no tendency to auto-reduce, and the reduced form is stable to air oxidation. Crystals suitable for X - r a y structure analysis were obtained from a concentrated solution of the protein (8 to 10 mg/ml) in 1.6 M-ammonium sulphate and 0.1 Msodium phosphate at p H 6-0. The protein solution was equilibrated with buffered a m m o n i u m sulphate solution at 4°C b y the v a p o r diffusion technique. The ammoninm sulphate concentration was raised from 1.6 M to 2"25 to 2"4 M during three to four weeks. The crystals which were formed under these conditions are thin olivegreen plates. S y m m e t r y and systematic absences on precession photographs show the space group to be P212121 with cell dimensions a = 30.8 A, b ---- 45.6 A, c = 66"6 A and V ---- 93,400 A a. The diffraction patterns extend to a resolution of at least 3 A. The crystals, like those of plastocyanin (Chapman et al., 1977), remain stable in the X - r a y beam for more t h a n one week. The partial specific volume inferred from the amino acid composition is 0.73 ml g - i ; the cell therefore contains four protein molecules which occupy 52% of the cell volume. The work was supported by grant 74/15398 from the Australian Research Grants Committee (to H. C. F.). One of the authors (P.M.C.) is a Queen Elizabeth I I Fellow. Department of Inorganic Chemistry University of Sydney Sydney 2006, Australia
PETER M. COLMAN H A N S C. F R E E M A N J. MITCHELL GUSS 1V/dTSUOMURATA VAX,ERIE A. NORRIS
JOHN A. M. RAMSHAW i~I.P. VEI~ATA~PPA~"
Department of Chemistry
LARRY E. VICKERY
University of California
Berkeley, Calif. 94720, U.S.A. Received 1 February 1977 REFERENCES Aikazyan, V. Ts. & Nalbandyan, R. M. (1975). P E B S Letters, 55, 272-274. Chapman, G. P., Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M., Norris, V. A., Ramshaw, J. A. M. & Venkatappa, M. P. (1977}. J. 111ol. Biol. 110, 187-189. Fee, J. A. (1975). Struct. Bond. 23, 1-60. Markossian, K. A., Aikazyan, V. Ts. & Nalbandyan, R. M. (1974). Biochim. Biophys. Acta, 359, 47-54. Peisach, J., Levine, W. G. & Blumberg, W. E. (19671. J. Biol. Chem. 242, 2847-2858. Vickery, L. E. (1971). Ph.D. Thesis, University of California, Santa Barbara. Vickery, L. E. & Purves, W. K. (1972). Plant Physiol. 49, 716-721. t Permanent address: Department of Chemistry, Karnatak University, Dharwar, India.
