Hemocyanin structure

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]. 

Ross, P. K., and E. I. Solomon. 1991. An Electronic Structural Comparison of Cooper-Peroxide Complexes of Relevance to Hemocyanin and Tyrosinase Active Sites. J. Am. Chem. Soc. 113, 3246. 

Senozan, N. et al. (1981) Hemocyanin of the giant keyhold limpet, Megathura crenulata. In Invertebrate Oxygen Binding Proteins Structure, Active Sites, andFunction (J. Lamy, and J. Lamy, eds.), pp. 703-717. Dekker, New York. 

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

The type 1-3 terminology to distinguish different Cu protein active sites remains extremely useful. Sub-groupings are appearing however in all three categories particularly in the case of the binuclear (EPR inactive) type 3 centers. Thus, in the recently determined X-ray crystal structure of ascorbate oxidase the type 3 and type 2 centers are present as a single trimer unit [. A discrete binuclear type 3 center is, however, retained in hemocyanin [6]. 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

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). 

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). 

The copper pairs are 42 A apart within a dimer, so that the coopera-tivity exhibited by this protein clearly involves indirect interactions. The fact that partially oxygenated hemocyanin can be found in the same crystal form (albeit with higher concentrations of dimethyl sulfoxide, which is a denaturant) argues that the structural changes either are not large or are within the variability already exhibited by the fact that the crystals at present cannot diffract to better than 3.2 A. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

Tire most studied of all copper-containing oxidases is cytochrome c oxidase of mitochondria. This multisubunit membrane-embedded enzyme accepts four electrons from cytochrome c and uses them to reduce 02 to 2 H20. It is also a proton pump. Its structure and functions are considered in Chapter 18. However, it is appropriate to mention here that the essential catalytic centers consist of two molecules of heme a (a and a3) and three Cu+ ions. In the fully oxidized enzyme two metal centers, one Cu2+ (of the two-copper center CuA) and one Fe3+ (heme a), can be detected by EPR spectroscopy. The other Cu2+ (CuB) and heme a3 exist as an EPR-silent exchange-coupled pair just as do the two copper ions of hemocyanin and of other type 3 binuclear copper centers. 

A number of the above complexes may be alternatively considered as macrocyclic ligands or compartmental ligands, but as the emphasis has been primarily in terms of the local copper(I) stereochemistry and the polynuclear nature of the complexes, they have been included above. As there is no crystallographic data on biological copper(I) systems, this section will have to await the further refinement of the structure of Panutirus Interruptus hemocyanin.353... 

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