Primary structure of the common polypeptide chain b from the multi‐hemoglobin system of the hydrothermal vent tube worm Riftia 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.

Evolution of Sulfur Binding by Hemoglobin in Siboglinidae (Annelida) with Special Reference to Bone-Eating Worms, Osedax

“…In contrast, the frenulate O. mashikoi possesses a single *400 kDA Hb composed of 24 globin chains with no linkers, comparable to the small extracellular Hbs of vestimentiferans (Yuasa et al 1996;Numoto et al 2005). Binding of H 2 S has been hypothesized to be mediated, in part, by cysteine residues in the V1 chains and by disulfide bridges formed from cysteine-rich linker chains (R. pachyptila's V1 chain b-B2 and L. luymesi's V1 chain AIII-A2; Zal et al 1996bZal et al , 1997. However, this only accounts for part of the binding affinity, and zinc moieties bound to amino acid residues at the interface between pairs of A2 chains may also be involved (Flores et al 2005).…”

“…For chain A2, a conserved-free cysteine at position 75, correlating to that found by Zal et al (1997), was present in all taxa except G. brachiosum (Fig. 2).…”

“…However, this only accounts for part of the binding affinity, and zinc moieties bound to amino acid residues at the interface between pairs of A2 chains may also be involved (Flores et al 2005). With reference to R. pachyptila's A2 chain, cysteines at positions 4 and 134 are common to all annelid globin chains studied and form a disulfide bridge while a free cysteine at position 75 is unique to sulfur oxidizing siboglinids (Zal et al 1997).…”

Metagenomics: Application of Genomics to Uncultured Microorganisms

“…The bacteria oxidize hydrogen sulfide, thereby producing the energy required to fix carbon from CO 2 , providing sugars and amino acids (predominantly as glutamate) that nourish the worm (55,84). The worm contributes to the symbiosis by collecting hydrogen sul- (3,46,(149)(150)(151)(152)(153). The bacterium is a member of the ␥-Proteobacteria, as identified by 16S rRNA gene sequence (47).…”

Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): First discovery of giant hexagonal-bilayer hemoglobin in the former “Pogonophora” group

“…The 400 kDa Hb are molecules measuring about one third the diameter of the HBL-Hb, looking like small circular rings on TEM microphotographs, and will therefore be named ring-Hb in the present study. In Riftia, all three hemoglobins are able to bind oxygen and sulfides, but none to such large amounts as the HBL-Hb (Zal et al, 1997) with 144 oxygen binding sites per protein. HBL-Hb binds both oxygen and sulfide reversibly, but at distinct sites of the molecule, avoiding both poisoning of the worm from free sulfides and spontaneous reaction between the two molecules (Arp et al, 1987;Zal et al, 1998).…”