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Hemocyanin is a complex oxygen carrier protein. It is found in the blue blood of some molluscs (e.g. octopus) and arthropods (e.g. crab). Unlike hemerythrin, which is found in cells, hemocyanin is an extracellular protein. It is put into the blood stream as a large aggregate of monomers held together by calcium or magnesium ions[1]. The structure of one deoxy-hemocyanin monomer from the giant octopus is shown on the left. It consists of 413 residues.
The monomers are organised in two domains: the N-terminal or core domain (light green) and the smaller C-terminal domain (violet).The N-domain consists of 313 amino acids in 12 α helices and eight short β strands. The final α helix connects the N-terminus with the C-terminus. The C-terminus has 100 residues in one α helix and 7 β strands.
The active site is located in the N-terminus near the domain interface. It contains two copper ions. They are both in the +1 oxidation state in deoxy-hemocyanin.
Three histidine residues coordinate each copper ion (labeled A and B). Note how the ligands come from the helices in the N-terminus and not its beta strands. This is an important fact that will be explained later. Each copper has a distorted trigonal pyramidal geometry with an empty cavity between them to accommodate a dioxygen molecule.
The two phenylalanine residues (Phe208, red and Phe66, yellow) are in close contact with the histidine ligands.They form a hydrophobic core that surrounds and protects the active site.
This is the active site of deoxy-hemocyanin. Apart from the already-mentioned histidine ligands and phenylalanine residues, this display shows a rather unusual thioether bridge between Cys59 and one of the histidine ligands. This connection seems to be of great importance.
This is the active site of oxy hemocyanin. When an O2 molecule coordinates to the active site, copper ions are oxidised to copper(II) and the protein changes from colourless to purple[2]. This change of oxidation state is accompanied by a change of geometry (from trigonal pyramidal to distorted tetrahedral) and by a change in ion size. These significant structural modifications of the active site are passed on to the surrounding alpha helices, in part via the thioether histidine-cysteine bridge. In this way the whole pocket in which the active site is located “cooperates” during the O2 binding.
These are two monomers of oxy-hemocyanin to explore further.
Before the high-resolution structures of hemocyanin became available, a lot of its properties and atypical spectroscopic properties (EPR and unusual UV spectra) have been understood through model compounds.
The structure of one of these model complexes is shown here [3]. This deep purple compound has two copper(II) ions bridged by a side-on bonded peroxide group. The additional ligand on both Cu centres is hydridotris[(3,5-di-isopropyl)-1-pyrazolyl]borate, [HB(C9H16N2)3]-.
This ligand, whose structure is presented here, plays the role of three histidine residues in the protein that connect the active site to the rest of the protein. It has been suggested that isopropyl groups mimic the two phenylalanine residues mentioned above.
The above dinuclear Cu(II,II) complex is prepared from a colourless mononuclear copper(I) complex Cu[HB(C9H16N2)3] in acetone under 1 atm of O2 at –78oC. The structure of this starting compound is not available yet.
However, the copper(I) complex also reacts with CO giving (CO)Cu[HB(C9H16N2)3]. Its structure is shown in the left frame.
If you would like to learn more about hemocyanin models see: