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The crystal structure of the oxygenated form of Limulus polyphemus subunit II hemocyanin.
20 ABSTRACTS
L16
Karen,
THE CRYSTAL STRUCTURE OF THE OXYGENATED FORM OF LIMULUS POLYPHEMUS SUBUNIT II HEMOCYANIN. and Hoa Ton-That
Department of Biochemism, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA
Hemocyanins are copper containing oxygen transport proteins found in arthropods and mollusks. Limulus polyphemus hemocyanin is a 48 protein subunit complex of 3.6 x 106 molecular weight consisting of eight subunit types. Each subunit, of approximately 73,000 daltons, can reversibly bind one oxygen molecule within its two copper active site. We have crystals of Limulus subunit type II suitable for x-ray diffraction studies. They have the,crystallographic symmetry R32 with lattice constants in the hexagonal setting of a = b = 117.2 f 0.6 and c = 286.9 f 0.9 A. There is one 73,000 dalton molecule per asymmetric unit’. We believe the protein to be in the oxygenated state due to the intense blue color of the crystals. Molecular replacement was used to obtain crystallographic phases2 found for the Limulus II crystals using the Panulirus interruptus hemocyanin model monomer as a test molecule structu@. Our current model of the Limzdus II structure is based on x-ray data to 2.4 A. It presently has an R-value of 19% and good stereo chemistry. The active site is clearly visible, each individual copper peak is about 20 standatd deviations above the noise level. The refmed distance between the two copper atoms is 3.4 f 0.2 A, which is significantly different from the copper to copper distance in deoxygenated crystals of Limulus II. Two extra electron density peaks are clearly distinguishable in the active site. By their spacing and locations we believe that they represent the two oxygen atoms of the molecular oxygen. The oxygen molecule is in the q?12conformation with regard to the copper atoms. There is no evidence in our maps for an exogenous bridging ligand. The closest non-protein electron density peak is within 4-5 A of the copper atoms. We postulate it is a water molecule or hydroxide ion based on its size and protein coordination. This work has been supported by NSF DNB90-04561. 1. Magnus, K.A. &Love, W.E. (1977) J. Mol. Biol. 116:171-173 2. Magnus, K.A. et al., (1991) Proteins 9240-247 3. Volbeda, A. & Hol, W.G.J. (1989) J. Mol. Biol. 209249-279
L16
Karen,
THE CRYSTAL STRUCTURE OF THE OXYGENATED FORM OF LIMULUS POLYPHEMUS SUBUNIT II HEMOCYANIN. and Hoa Ton-That
Department of Biochemism, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA
Hemocyanins are copper containing oxygen transport proteins found in arthropods and mollusks. Limulus polyphemus hemocyanin is a 48 protein subunit complex of 3.6 x 106 molecular weight consisting of eight subunit types. Each subunit, of approximately 73,000 daltons, can reversibly bind one oxygen molecule within its two copper active site. We have crystals of Limulus subunit type II suitable for x-ray diffraction studies. They have the,crystallographic symmetry R32 with lattice constants in the hexagonal setting of a = b = 117.2 f 0.6 and c = 286.9 f 0.9 A. There is one 73,000 dalton molecule per asymmetric unit’. We believe the protein to be in the oxygenated state due to the intense blue color of the crystals. Molecular replacement was used to obtain crystallographic phases2 found for the Limulus II crystals using the Panulirus interruptus hemocyanin model monomer as a test molecule structu@. Our current model of the Limzdus II structure is based on x-ray data to 2.4 A. It presently has an R-value of 19% and good stereo chemistry. The active site is clearly visible, each individual copper peak is about 20 standatd deviations above the noise level. The refmed distance between the two copper atoms is 3.4 f 0.2 A, which is significantly different from the copper to copper distance in deoxygenated crystals of Limulus II. Two extra electron density peaks are clearly distinguishable in the active site. By their spacing and locations we believe that they represent the two oxygen atoms of the molecular oxygen. The oxygen molecule is in the q?12conformation with regard to the copper atoms. There is no evidence in our maps for an exogenous bridging ligand. The closest non-protein electron density peak is within 4-5 A of the copper atoms. We postulate it is a water molecule or hydroxide ion based on its size and protein coordination. This work has been supported by NSF DNB90-04561. 1. Magnus, K.A. &Love, W.E. (1977) J. Mol. Biol. 116:171-173 2. Magnus, K.A. et al., (1991) Proteins 9240-247 3. Volbeda, A. & Hol, W.G.J. (1989) J. Mol. Biol. 209249-279