After the evolution of the first pluricellular organisms, Nature has provided three main types of oxygen transporters present in the living organisms: Hemoglobin (Hb), Hemerythrin (Hr) and Hemocyanin (Hc). Hemocyanin is known as the respiratory pigment present in several mollusks and arthropods that allows the caption and further use of oxygen. This is possible due to the presence of di-copper center in the active site (Type-3 copper protein), at the inner part of the protein. Each Cu atom is surrounded by three histidines (His) and each active site can reversibly bind one molecule of O2 forming an unusual peroxide bridge. Due to this reversible binding there is a transition between two different states of the protein: Deoxy-Hc and Oxy-Hc, which is performed through a redox reaction where the Cu(I) of Deoxy-Hc is oxidized to Cu(II) (Oxy-Hc) and O2 is reduced to O22-. Each state offers enough characteristic features to be easily identified. Recently, we have focused on the Hc of Cantareus aspersum. In this Gastropoda species, Hc consists on a cylindrical didecamer with a molecular mass of 8000 kDa. Each decamer is composed by five dimers of subunits, and each subunit, which has a molecular mass of 400 kDa, is made of 8 functional units (FUs). Each FU contains one di-copper center, able to bind one O2 molecule. Gastropoda, and specially snails, have two metallothionein (MT) isoforms: a CdMT which is Cd-specific, and a CuMT for Cu. Several literature studies suggest that Hc and CuMT are synthesized in the rhogocyte cells and this has given rise to the hypothesis that there can be a relationship between Hc and CuMT in which the latter could be the source of Cu(I) for the nascent Hc. The main objective of this project is to ascertain the involvement of Cu-specific metallothioneins (CuMTs) as the source of Cu(I) in the synthesis of Hemocyanin (Hc) in the snail Cantareus aspersum. To achieve this goal, several analytical techniques have been used to characterize the behavior, structuration and composition of the different preparations: UV-Vis spectroscopy, fluorescence spectroscopy, SEC, TEM and ICP-OES. Moreover, several experiments have been designed with the aim of i) obtaining the apo-Hc form and studying its reconstitution process, first through a direct transfer of Cu(I) ions and afterwards, with a Cu(I) transfer from the CuMT, and ii) determining how the different life stages of the snails and their diet affect the Hc status of these organisms.
Hemocyanin's study from a chemical point of view
Pons Garcia, A. (Author). 15 Mar 2024
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis