© 2005 The Japanese Biochemical Society
Regular Paper |
Structure of Hemocyanin Subunit CaeSS2 of the Crustacean Mediterranean Crab Carcinus aestuarii
1 Institute of Organic Chemistry, Bulgarian Academy of Sciences, G. Bonchev 9, Sofia 1113, Bulgaria; 2 Abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der Universität Tübingen, Hoppe-Seyler-Straße 4, 72076 Tübingen, Germany; 3 Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium; 4 Anorganische Biochemie, Physiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Straße 4, 72076 Tübingen, Germany; 5 Department of Biology and CNR Institute for Biomedical Technologies, Section of Padova, University of Padova, Via Ugo Bassi 58/B, I-35131 Padova, Italy; and 6 Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany
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Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the 
-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex CuII(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.