ORIGINAL ARTICLE A. Sabatucci á I. Ascone á L. Bubacco á M. Beltramini P. Di Muro á B. Salvato Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins Received: 15 November 2000 /Accepted: 3 June 2001 / Published online: 4 August 2001 Ó SBIC 2001 Abstract The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge XANES) and for the ®rst shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla dierences. The XANES spectra of oxy-hemocyanins of the dierent species are re- markably similar, consistent with a very strongly con- served co-ordination geometry of the copper active site. In contrast, small but signi®cant dierences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy- arthropodan hemocyanins with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This dierence is tentatively assigned to a more planar geometry of the copper-li- gands system in the arthropodan rather than in the molluscan proteins. The ®rst shell analysis of the EXAFS modulation is consistent with the presence of n=3N 2 imidazole ni- trogens at an average distance of 1.920.03 A Ê from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of ®rst shell back-scattering at- oms to n=5 with average distances of 1.93 A Ê . Alterna- tively, by separating the contribution of N 2 imidazole nitrogens and of peroxide O-atoms, n=3 ligands at 1.980.03 A Ê and n=2 ligands at 1.870.03 A Ê are found. Keywords Hemocyanins á X-ray absorption spectroscopy á Dinuclear copper site Introduction Hemocyanins Hcs) are the oxygen transport/storage copper proteins found in the hemolymph of several species belonging to the phyla of mollusks and arthro- pods. Their biological function is based on the reversible binding of molecular dioxygen to a dinuclear copper active site. This binding involves a metal-to-ligand electron transfer from the two CuI) ions present in the deoxy-Hcs to dioxygen to generate, in the oxygenated form, a dinuclear CuII)-peroxide complex where the di-anion is bound in a l:g 2 -g 2 bridging mode [1, 2]. X-ray structures have been provided for two deoxy- Hc forms, from the lobster Panulirus interruptus and the horseshoe crab Limulus polyphemus, and two oxy-Hc forms, from Limulus polyphemus and the octopod Octopus vulgaris. In all cases, each copper ion Cu A , Cu B ) is bound to three histidine residues. As expected, the topologies of the two arthropodan Hcs are essen- tially the same. Arthropodan Hcs are folded to form a three-domain structure. Domain 1, in the N-terminal region, is mainly folded in a-helices. Domain 2, like domain 1, is mainly helical and globular. This region is deeply buried into the protein matrix and presents a marked hydrophobic character. Finally, the C-terminal domain 3 is the largest one and its core is a seven- stranded b-barrel structure. The active site is located in domain 2. Each copper ion is co-ordinated by three conserved histidine residues. J Biol Inorg Chem 2002) 7: 120±128 DOI 10.1007/s007750100272 Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/ s007750100272. I. Ascone LURE, CNRS) Centre Universitaire Paris XI, baÃt. 209D, B.P. 34, 91898, Orsay Cedex, France A. Sabatucci á L. Bubacco á P.D. Muro Dipartimento di Biologia, UniversitaÁ degli studi di Padova, via U. Bassi, 58/B, 35121, Padova, Italy B. Salvato &) á M. Beltramini Dipartimento di Biologia, Centro CNR emocianine, UniversitaÁ degli studi di Padova, via U. Bassi, 58/B, 35121, Padova, Italy E-mail: salvato@civ.bio.unipd.it Present address: A. Sabatucci Sezione di Biochimica e Biologia Molecolare, FacoltaÁ di Medicina Veterinaria, UniversitaÁ di Teramo, Loc. Piano d'Accio, 64020 Nepezzano TE), Italy