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

Abstract

Key to the remarkable ability of vestimentiferan tubeworms to thrive in the harsh conditions of hydrothermal vents are hemoglobins that permit the sequestration and delivery of hydrogen sulfide and oxygen to chemoautotrophic bacteria. Here, we demonstrate that zinc ions, not free cysteine residues, bind sulfide in vestimentiferan hemoglobins. The crystal structure of the C1 hemoglobin from the hydrothermal vent tubeworm Riftia pachyptila has been determined to 3.15 A and revealed the unexpected presence of 12 tightly bound Zn(2+) ions near the threefold axes of this D(3) symmetric hollow sphere. Chelation experiments on R. pachyptila whole-coelomic fluid and purified hemoglobins reveal a role for Zn(2+) ions in sulfide binding. Free cysteine residues, previously proposed as sulfide-binding sites in vestimentiferan hemoglobins, are found buried in surprisingly hydrophobic pockets below the surface of the R. pachyptila C1 molecule, suggesting that access of these residues to environmental sulfide is restricted. Attempts to reduce the sulfide-binding capacities of R. pachyptila hemoglobins by addition of a thiol inhibitor were also unsuccessful. These findings challenge the currently accepted paradigm of annelid hemoglobin evolution and adaptation to reducing environments.

Fig. 1.

Six-fold-averaged electron density map for the R. pachyptila C1 Hb. The electron density map derived from phase extension by using molecular averaging of the six A1A2B1B2 tetramers in R. pachyptila C1 is shown for the heme region of the A2 subunit contoured at 3 σ (light blue) and 15 σ (purple). The quality of this 3.15-Å map is excellent and allows confident placement of side chains and clear definition of the heme iron position.

Fig. 2.

Zn²⁺-binding sites. (Left and Center) Stereoview of two Zn²⁺-binding sites with protein ligands along with a 2Fo – Fc map contoured at 6 σ. Each A2 subunit provides one complete Zn²⁺-binding site involving three His ligands and shares two others at interfaces with neighboring A2 subunits. The refined Zn²⁺ positions are shown as purple balls. (Right) The arrangement of Zn²⁺-binding sites near the molecular threefold axis of R. pachyptila C1 Hb. Zn²⁺ ions are purple, along with protein ligands and main-chain traces in the vicinity of the Zn²⁺ ions.

Fig. 3.

Overview of the R. pachyptila C1 Hb structure. The subunit trace for A1 is light green, A2 is dark green, B1 is light blue, and B2 is dark blue. In addition, the heme groups are red, the Zn²⁺ ions are purple, free-Cys residues are yellow, and the protein side-chain ligands are dark green. (Upper) The view along the threefold axis. (Lower) The view along the twofold axis. Note the location of the Zn²⁺ atoms clustered in groups of six near the threefold axis at both poles of the molecule.

Fig. 4.

Stereoviews of the hydrophobic pockets surrounding the two free-Cys residues that are proposed as sites of sulfide binding. (Upper) Cys E18 in subunit A2. (Lower) Cys E8 in subunit B2. In both cases, the free-Cys is buried ≈5 Å beneath the protein surface in a hydrophobic pocket, with a nearby (≈5.5 Å) glutamine residue.