Sulphide-binding processes of Riftia pachyptila haemoglobins

found around East-Pacific Rise hydrothermal vents is the giant tube-worm Riftia pachyptila Jones, 1981. This “autotrophic” animal is devoid of a digestive system and acquires metabolic energy from the oxidation of sulphide by chemolithoautotrophic bacteria that live symbiotically inside a specific, highly vascularized organ, the trophosome. Therefore, blood transport nutrients is a crucial issue for this animal (review in Childress & Fisher, 1992). Riftia supplies its internal symbionts with oxygen and sulphide via extracellular haemoglobins (Hbs), two dissolved in the vascular blood (V1 and V2) and one in the coelomic fluid (C1) (Zal et al., 1996a) (Fig. 1). Indeed, Riftia’s Hbs possess an unusual property to bind simultaneously and reversibly oxygen and sulphide on two different sites (Arp et al., 1987). However, in spite of this singular property, the V1 Hb exhibits the typical hexagonal bilayer structure commonly found in annelids (see Toulmond & Truchot, 1993), comprising a total of 180 polypeptide chains with 144 globin chains and 36 linker chains of four different types (Fig. 1 A). The smaller Hbs, V2 and C1, consist only of 24 globin-like chains (Zal et al., 1996a) (Fig. 1 B). Interestingly, as for other Vestimentifera and Pogonophora, there are free cysteine (Cys) residues on two globin-chains common to V1, V2 and C1 (Zal et al., 1996b). Although, free Cys residues have been proposed previously as the sulphide-binding sites of the Hbs in these symbiotic species (Arp et al., 1987), this hypothesis has never been verified. Ten years after the discovery of Riftia’s Hbs ability to bind sulphide (Arp et al., 1987), we have now identified the sites involved in this unusual mechanism (Zal et al., 1997, 1998). Indeed, recent findings on vertebrate Hbs may help understand the mechanism of sulphide-binding by Riftia Hb. Jia et al. (1996) reported the responsibility of the reactive thiol group at position ß93 on mammals and birds ß globin chains. These authors showed that S-nitrosothiol groups (SNO) could bind reversibly on this reactive sulfydryl (SH) forming the so-called S-nitrosohaemoglobin (Fig. 2 A). Similarly we showed recently that reactive thiol groups, highlighted on two globins from Riftia’s Hbs, bind sulphide, likely following a similar mechanism forming the so-called S-sulfohaemoglobin by analogy with the term mentioned above (Figs. 2 B, 3 A) (Zal et al., 1997, 1998). In addition, recent results on stoichiometry between sulphide binding versus Cys contained on Riftia’s Hbs and sulphide-binding inhibition experiments, showed that free Cys on globin-like chains and Cys involved in disulphide bridges on linker chains were responsible for the sulphide binding properties of Riftia Hbs (Zal et al., 1997, 1998) (Figs. 3 A, B). Indeed, only the participation of linker chains in sulphide binding could explain the observed difference between the binding capacity of V1 Hb and that of V2 or C1 Hbs, since these three Hbs are built with almost the same globin components (Zal et al., 1996a). Cah. Biol. Mar. (1998) 39 : 327-328