Limulus hemocyanin

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). 

The stability of the black copper complex and its tendency to form soluble, highly colored complexes with amines (and also denatured albumen) indicates that the compound we have in hand is involved in the union of the copper to the protein in hemocyanin. The function of the prosthetic group thus appears to be the same as that of protoporphyrin in hemoglobin, namely, to provide a basis for a very stable metallic complex. Beyond this analogy, however, there seems to be little or no chemical relationship between the prosthetic groups in limulus hemocyanin and hemoglobin. It should be noted that since limulus hemocyanin differs markedly in its copper content from the hemocyanin of other species,2 the conclusions we have drawn do not necessarily apply to the nature of the prosthetic group in the other hemocyanins. 

Experiments with Busycotypus hemocyanin yielded results similar to those obtained with Limulus hemocyanin. However, with an (OD)0 of 0.5, radiation doses above 13,000 to 16,000 rads restored the oxygen capacity, and above 70,000 rads the oxygen capacity became nearly equivalent to unirradiated hemocyanin (37). When the concentration of hemocyanin was increased to give an (OD) 0 of 0.9, the radiation dose required to produce the minimum increased to that expected—observed... 

Effect of Organic Peroxides. The effect on the oxygen-carrying properties of Limulus hemocyanin by different organic peroxides was tested. The peroxides tested included succinic acid peroxide (HOOC— CH2—CH2—C0)202 tert-butyl hydroperoxide ((CH3)aC—OOH) and... 

Figure 6. Liberation of oxygen from hydrogen peroxide in the presence of Busycotypus hemocyanin at pH 7 but at different temperatures and of Limulus hemocyanin at 25°C. and pH 9.5. The Busycotypus hemocyanin solutions were heated for 15 minutes at a given temperature prior to mixing with...

Discrepancies exist in the literature about the reaction of hemocyanin with carbon monoxide. It has been known for a long time that, in contrast to oxyhemocyanin which is blue, solutions of hemocyanin treated with CO are colorless (Craifalenau, 1919). Root (1934) showed that Limulus hemocyanin combines with carbon monoxide in proportions similar to those of oxygen. However, besides the loss of color, the affinity of this hemocyanin for carbon monoxide is only one-twentieth of that for oxygen. In contrast with these results, Rawlinson (1940) found no evidence for combination with carbon monoxide by hemocyanin from Palinurus vulgaris. 

Figure 7 of reference 11 compares similarities between Limulus and Octopus hemocyanins by illustrating the overlap of 30 amino acids about the CuB region. The authors find that all residues within 5.0 A of the Cu-Cu midpoint are conserved between arthropodan and molluscan hemocyanins and occupy analogous positions except for one (see the following paragraph). The three histidines about CuB overlap almost exactly in orientation and are less than 1 A apart. The histidines... 

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). 

Both cations play a stabilizing role in various biological structures. Calcium ions are necessary to link the 24-subunit aggregates of the dioxygen carrier hemocyanin of Limulus polyphemus into the native 48-subunit molecule.121 The divalent character of Ca2+ probably allows it to crosslink... 

The deoxy forms of hemocyanins are colorless, as a result of their 3d ° dicopper(I) centers. Although chemical and x-ray absorption spectroscopic studies had shed considerable light on the nature of the deoxy-He dicopper binding site, there now exist two x-ray crystal structures, the first on the the spiny lobster He, Panulirus interruptus [23], and a recent one of the horseshoe crab Limulus II protein [24], The structures exhibit rather different active-site characteristics, and since the former was crystallized at low pH and possesses rather odd copper coordination, the latter Limulus II structure is probably representative. It indicates that the two Cu(I) ions are 4.6 A apart, each found in a trigonal-planar coordination environment with Cu-NMs bond distances of about 2.0 A (Figure 1). Intersubunit 02 binding cooperative effects are probably initiated and trans-... 

Figure 1 Oxy (a) and deoxy (b) states of the active sites of subunit II of Limulus polyphemus hemocyanin. The three histidines coordinating Cu-A are colored red, those coordinating Cu-B green. The two copper atoms are colored blue and oxygen red. Yellow lines connect the four histidines forming the plane on which the coppers and the dioxygen are often discussed to be placed. Two axial histidines are perpendicularly oriented to this plane His204 coordinates the Cu-A site, His328 the Cu-B site. The channel to the surface for substrates to the active sites is located on the top of the active sites...

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)...

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 ...

Fig. 7. The type 3 copper center of hemocyanin from Limulus polyphemus. From Hazes et al. 1993 [37] with permission. This figure and Fig. 30 were based on the Art Blot Program [38]...

The hemocyanin subunits from Limulus polyphemus are also subdivided into three domains, which consist of the amino acids 1-154 (domain 1), 155-380 (domain 2), and 381-628 (domain 3) (Fig. 29). Domains 1 and 2 are primarily a-helical, whereas domain 3 consists mainly of /(-strands. Domain 2 is situated between the other two domains and contains the copper center. Each copper is coordinated by three histidine residues, which come from two helices. Subunit 2 is primarily responsible for the contact between the hexamer subunits. The chloride-binding site is situated between domains 1 and 2, and the calcium-bin-ding site is located in domain 3, which has a seven-ribbon /(-meander structure [37],... 

Brouwer M, Bonaventura C, Bonaventura J (1982) Chloride and pH dependence of cooperative interactions in Limulus polyphemus hemocyanin. In Bonaventura J, Bonaventura C, Tesh S (eds) Physiology and biology of horseshoe crabs studies on normal and environmentally stressed animals. Alan R Liss, New York, p 231... 

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