Primary structure of the common polypeptide chainb from the multi-hemoglobin system of the hydrothermal vent tube wormRiftia pachyptila: An insight on the sulfide binding-site

Abstract

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.

The Evolution of Extracellular Hemoglobins of Annelids, Vestimentiferans, and Pogonophorans

“…The evolutionary pathway does not change even if we consider pogonophorans and vestimentiferans as members of the class Opisthochaeta within the phylum Annelida (20 …”

“…These authors proposed the division of the globin chains into two main groups, A and B, each divided further into two subgroups, A1/A2 and B1/B2. This classification was successively applied to Hb H s and Hb L s (19,20). Recently, a correction of this nomenclature has been proposed that recommends an inversion of the names of the two main strains (21).…”

Sulfide binding is mediated by zinc ions discovered in the crystal structure of a hydrothermal vent tubeworm hemoglobin

“…A molecular model for the R. pachyptila C1 Hb was fit in O (16) by using the published sequence for A2 (8), partial sequences for the A1, B1, and B2 chains (17), and previously unpublished terminal amino acid sequences determined by nucleotide sequencing of R. pachyptila genomic DNA (see Table 3, which is published as supporting information on the PNAS web site).…”

“…Sulfide binding to these Hbs has been thought to involve the numerous cysteine residues (both free and disulfide bonded) found in the polypeptide chains of these Hbs (8)(9)(10). Specifically, free cysteine (free-Cys) residues not involved in disulfide bonds have been proposed as the sole location of sulfide binding in the 400-kDa (V2 and C1) Hbs, whereas it has been hypothesized that the V1 Hb has multiple sulfide-binding mechanisms involving free-Cys residues and the abundant intrachain disulfide bonds in the nonglobin linker chains (8,9).…”

The Primary Structure of Globin and Linker Chains from the Chlorocruorin of the Polychaete Sabella spallanzanii

“…Five cysteines, crucial for the subunit formation, are present. Two adjacent cysteines just preceding A1 and one at position H11 are conserved as in all annelid globin chains of type I (18). Two other cysteines occur at position E8 and within the corner between the G and H helices, as frequently seen in other annelid Hbs as well (19).…”