Entrapment and characterization of functional allosteric conformers of hemocyanin in sol–gel matrices

Hemocyanins are giant oxygen transport proteins of molluscs and arthropods, which display high cooperativity and a complex pattern of conformations, generated by hierarchical allosteric interactions of their complex quaternary structure. A still unanswered question is the correlation between the functional properties of the postulated conformers and structural features that govern their oxygen binding, such as metal complex coordination. In this study we focus on the dodecameric hemocyanin of the crustacean Carcinus aestuarii, with the aim to obtain a functional and structural characterization of the individual conformational states giving rise to cooperativity, by entrapping hemocyanin into a sol–gel matrix. The latter has attracted much attention as an ideal substrate for immobilization of macromolecules within the pores of a hydrated and optically transparent matrix that preserves the structures and functionalities of the encapsulated macromolecules. In our experimental approach, the sol–gel is capable of blocking the conformational transitions of the hemocyanin induced by changing the oxygen concentration in solution studies. This enables characterization of both the oxygenated and deoxygenated forms of particular conformers. Here we describe the oxygen binding properties of individual matrix entrapped conformers of C. aestuarii hemocyanins and the spectroscopic features characteristic for these conformations. Since the quaternary structure itself is not altered, as the SANS data unambiguously show in the sol–gel a dodecameric organization for C. aestuarii hemocyanin, we propose that the entrapment of hemocyanin in a sol–gel is able to freeze functionally relevant conformational distributions under appropriate conditions. The spectroscopic characterization of the functionally characterized conformers trapped in the sol gel allowed us to assign the differences in the active site geometry as observed by XAS to individual conformations.