The zinc-mediated sulfide-binding mechanism of hydrothermal vent tubeworm 400-kDa hemoglobin

Abstract

Hydrothermal vent and cold seep tubeworms possess two hemoglobin (Hb) types, a 3600-kDa hexagonal bilayer Hb as well as a 400-kDa spherical Hb. Both Hbs can reversibly and simultaneously bind and transport oxygen and hydrogen sulfide used by the worm's endosymbiotic bacteria to fix carbon. The vestimentiferan 400-kDa Hb has been shown to consist of 24 polypeptide chains and 12 zinc ions that are bound to specific amino acids within the six A2 globin chains of the molecule. Flores et al. (2005) determined that the ligated zinc ions were directly involved in the sulfide binding mechanism of this Hb. This discovery contradicted previous work suggesting that free-cysteine residues were the sole sulfide binding mechanism of the 400-kDa Hb. In the present study, we investigated the effects of acidic pH pretreatment and zinc chelator concentrations on the binding of sulfide by the Hb. We show that acidic pH pretreatment, as well as NEM capping of free-cysteines, does not affect sulfide binding by the purified Hb. In addition, complete inhibition of sulfide binding was reached when the concentration of the zinc chelator, TPEN, was increased compared to the initial study. These results demonstrate the essential role of zinc in sulfide binding by the 400-kDa Hb from Riftia pachyptila.