Heavy metal ion interactions with Callinectes sapidus hemocyanin: structural and functional changes induced by a variety of heavy metal ions

Publisher Website
Google Scholar
Abstract
Hemocyanins are oligomeric proteins that reversibly bind O2. The O2 binding site is a binuclear Cu center bound to the protein by amino acid side chains. The hemocyanin of the blue crab, C. sapidus, occurs in vivo as a mixture of 25S dodecamers and 16S hexamers, whose O2 binding properties are identical. Four heavy metals were used as probes of structure and function in this hemocyanin system. Divalent cations of Cd, Cu, Hg and Zn induced an indefinite self-association of the hemocyanin molecule. These higher ordered association states can be dissociated by ethylenediaminetetraacetic acid. Callinectes oxyhemocyanin possesses at least 3 Hg binding sites: a sulfhydryl group which forms a mercaptide bond with a single mercuric ion, a tryptophanyl side chain which forms a noncovalent 1:1 complex with mercuric ions with an association constant of 5.7 .times. 1015 M-1 and lower affinity site(s) involved in the self-association process also observed with Cd, Cu and Zn. Sites 1 and 2 are most likely also involved in the binding of Cd. Upon removal of O2 from the active site of hemocyanin, an additional binding site becomes available for the reaction with Hg. Binding of mercury to this site leads to loss of 1 of the Cu from the binuclear O2 binding site. Both the binuclear Cu center and allosteric sites on the hemocyanin are affected by heavy metal binding. Cd and Zn ions increase the O2 affinity; Hg and Cu ions have the opposite effect. All 4 heavy metal ions decrease the degree of cooperative O2 binding. The Hg induced changes in O2 binding by 25S Callinectes hemocyanin appear to be the result of that metal''s interaction with the high-affinity tryptophan binding site. Hg binding to the available sulfhydryl group in oxyhemocyanin occurs without functional consequences. Heavy metal, H and Cl ions affect the affinity of the 1st or last O2 molecules bound to the hemocyanin, which results in the appearance of multiple T (low O2 affinity) and R (high O2 affinity) states. These ions also shift the equilibrium between the low and high O2 affinity states. The appearance of additional R states at high pH is accompanied by the cleavage of a tyrosine hydrogen bond.