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

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemog lobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca, 400 kDa), and one i n the coelomic fluid, C1 (ca, 400 kDa). VI Hb consists of four heme-co ntaining, globin chains (b-e) and four linker chains (L1-L4), V2 and C 1 Hbs are exclusively built from globin chains, six for V2 (a-f) and f ive for C1 (a-e). The complete amino acid sequence of the isolated mon omeric globin chain b, common to all Riftia Hbs, has been determined b y automated Edman degradation sequencing of the peptides derived by di gestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polype ptide chain is composed of 144 amino acid residues, providing a Mr 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 anne lid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lame llibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosyntheti c process of the symbiotic bacteria they harbor, Sulfide-binding exper iments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) s uggest that free Cys residues on globin chains, and the numerous Cys f ound in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites, Blocking the free Cys by N-ethylmaleimide, we c onfirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermo re, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vetimentiferan, 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 sequ ences analyzed, Its position in the tree confirmed a close relationshi p between vestimentiferan, pogonophoran, and annelid Hbs. (C) 1997 Wil ey-Liss, Inc.