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The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a M(r) of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vestimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the "strain A" family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs. |
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