Arthropodal hemocyanins

The arthropod hemocyanins have yielded the most to diffraction studies. Studies of Limulus hemocyanin by Magnus and Love (1983, cited by Preaux and Gielens, 1984) showed a kidney bean-shaped subunit which is consistent with both electron micrographs and the structure of hemocyanin from Panuliris interruptus (spiny lobster). 

An attempt is being made to find crystals of an arthropod hemocyanin which diffract to high resolution and have identical subunits in the asymmetric unit. To that end Buisson et al. (1989) crystallized subunit Aa6 of Androctonus australie hemocyanin in two forms one which diffracts only to 8 A and one which diffracts to 3 A. 

By far the most definitive study on an arthropod hemocyanin has been that by Volbeda and Hoi (1989b), a crystallographic tour de force. Crystals of subunit b can be formed from solutions of native hemocyanin which contain three types of subunits (a, b, and c). Two subunits (a and b) are nearly identical (3% difference in sequence), whereas subunit c differs more. Subunits a and b are glycosylated at a single residue (Asn-167). While the Panuliris form has been shown to be deoxy (Volbeda et al., 1989), unpublished observations indicate that the horseshoe crab structure Limulus) is in the oxygenated state (K. Magnus, personal communication, 1988, cited by Volbeda and Hoi, 1988). 

Burmester, T. (2001). Molecular evolution of the arthropod hemocyanin superfamily. Molecular Biology <6 Evolution 18 184-195. 

Burmester, T. and K. Scheller (1996). Common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin, and dipeteran arylphorin receptor. J. Mol. Evol. 42 713-728. 

Figure 5 Comparison of the tertiary structures of a catecholoxidase from a sweet potato Ipomea sp. (a), functional units of molluscau hemocyauins (O. dofleini (b) and R. thomasiana (c)) and arthropod hemocyanin subunits from L. polyphemus (d) and P. interruptm (e). All structures are centered with respect to domain 11 (red) carrying the active site, domain 1 is colored green, domain 111 blue. The histidines are colored gray. In the Limulus structure (d) the alpha 1,3 helix is missing compared to the Panulirus (e) structure (ellipse)...

Comparing the most conserved parts, the active site, reveals two different types of type 3 copper proteins, an arthropod hemocyanin-like one and a molluscan hemocyanin-hke one. While the Cn-B site is highly conserved in both types, the Cu-A site differs. In the case of molluscan hemocyanins, one helix is too short for stabihzing the important histidine complexing Cu-A so that it has to be tied down by an unusual His ys bond as shown in Figure 2 (d-f). 

A similar substrate-binding pocket was identified for molluscan hemocyanins, which was not surprising since the active site of mollusc hemocyanins and that of arthropod hemocyanins and catecholoxidases are very similar. Therefore, we proposed the following mechanism for the activation of the molluscan hemocyanin based on the recently solved X-ray structure of a fimctional unit of the hemocyanin from the molluscan O. dofleini The C-terminal domain covers the entrance to the active site located on the N-terminal site by sticking Leu2830 into the entrance door. Detergents... 

Figure 8 Proposed mechanism of phenoloxidase activity in arthropod hemocyanins based on two X-ray structures of the oxy- and deoxy-forms of the Limulus submit II ...

Evolution of two phenoloxidases, an arthropod and molluscan type. A close relationship between phenoloxidase and hemocyantn was deduced based on their similar sequences, physico-chemical properties and similar functions. But sequence comparisons also revealed that there is not a common phenoloxidase type the enzymes found in animals, plants, and fungi are different with respect to their sequences, size, glycosylation, and activation. Two different types of tyrosinases can be distinguished based on their sequences, structure, and function. One type (m-phenoloxidase) is more related to molluscan hemocyanin with respect to the active site. The other type (a-phenoloxidase), which is very similar to arthropod hemocyanins, is found in arthropods together with hemocyanins (Figure 9). ... 

Comparison of the arrangements of the domains within an arthropod hemocyanin subunit and the functional unit of a molluscan hemocyanin also suggests two different types of phenoloxidases, the a-phenoloxidase and the m-phenoloxidase. Cleavages of the N-terminal domain of a-phenoloxidase and the C-terminal domain of m-phenoloxidase open the entrances to the active sites for bulky substrates. The key amino acids are indicated in Figure 9. 

Contrary to the results for molluscan hemocyanin, arthropod hemocyanin does not undergo rapid aging upon fluoride treatment as evidenced by the lack of change in the optical spectrum84. However, an interaction of F- and arthropod oxyhemocyanin was observed by NMR studies82 (vide ante) and the maintenance of the 340 nm absorption is due to the retention of 02 by the oxyhemocyanin in spite of the presence ofF". 

After H202 oxidation, arthropod hemocyanin also reacts with N3 and F- to produce a magnetically tightly coupled product86. But inasmuch as H2 02 oxidation produces a diamagnetic nonreducible methemocyanin the anion interaction does not seem surprising or easily interpreted at this time. 

A comparison of the CD spectra in the range 260—700 nm of mollus-can and arthropod hemocyanins showed large differences which were attributed to fundamental differences in the oxygen binding sites of these... 

The hemocyanins are the oxygen-transport proteins of the three largest classes of mollusks (e.g., snails and squids), and of arthropods (e.g., spiders and scorpions). Mollusk and arthropod hemocyanin resemble each other in function... 

Arthropod hemocyanins (A-Hc) are proteins with molecular masses of up to 450 kD. They may be dissociated into six functional subunits of 75 kD mass, each of which contains a binuclear type 3 copper center responsible for oxygen binding. These proteins are, consequently, hexamers or multiple units thereof, which occur as native aggregates of 1 x 6,2 x 6,4 x 6, and 8x6 subunits. The latter have molecular masses of 3600 kD. The spider Eurypelma californicum possesses a hemocyanin structure of 4x6 [34]. These 24 subunits maybe classified into 7 different types a,b,c,d,e,f, and g, of which subunits a,d,e,f, and g occur 4 times, and the subunits b and c twice [236]. Each subunit has a specific position within the structure of the protein. Each protein subunit, i.e., the oxygen-binding unit, consists of three domains. Domains 1 (175 amino acids) and 2 (230 amino acids) have a pronounced a-helical structure, whereas domain 3 (250 amino acids) consist almost completely of /(-strands, which are arranged in a /(-barrel structure similar to that of Cu,Zn-SOD [34]. 

The function of domain 1 has not yet been completely elucidated, although it may function as a medium of cooperative oxygen binding [37]. Domain 3 screens the copper center of domain 2 [237] from solvent. This copper center in domain 2 [34] is coordinated by six histidines from the helices 2.1 (2 His), 2.5 (2 His), 2.2 (1 His), and 2.6 (1 His). The histidine residue of helix 2.1 occurs in the sequence His-His-Trp-His-Trp-His, which is conserved in many arthropod hemocyanins. The histidines in helix 2.5 occur in the structure His-X-X-X-His. The coordination of two copper atoms with six ligands leaves two coordination sites free these two sites are utilized to bind oxygen [34],... 

The active centers of tyrosinase and hemocyanins are very similar [254], although they fulfill very different functions. In both proteins, a binuclear center is coordinated by six histidines, leaving two coordination sites free to bind oxygen [237], The main difference in the active centers of the proteins is their position within the protein. In arthropod hemocyanin, the copper center is located in domain 2 of every subunit. Domain 3 folds over the copper center, rendering it inaccessible to substrates larger than oxygen [237]. In tyrosinase, a C-terminal peptide of 200 amino acid residues is removed by post-translational... 

---