Structure of an extracellular giant hemoglobin of the gutless beard worm Oligobrachia mashikoi

Abstract

Mouthless and gutless marine animals, pogonophorans and vestimentiferans, obtain their nutrition solely from their symbiotic chemoautotrophic sulfur-oxidizing bacteria. These animals have sulfide-binding 400-kDa and/or 3,500-kDa Hb, which transports oxygen and sulfide simultaneously. The symbiotic bacteria are supplied with sulfide by Hb of the host animal and use it to provide carbon compounds. Here, we report the crystal structure of a 400-kDa Hb from pogonophoran Oligobrachia mashikoi at 2.85-A resolution. The structure is hollow-spherical, composed of a total of 24 globins as a dimer of dodecamer. This dodecameric assemblage would be a fundamental structural unit of both 400-kDa and 3,500-kDa Hbs. The structure of the mercury derivative used for phasing provides insights into the sulfide-binding mechanism. The mercury compounds bound to all free Cys residues that have been expected as sulfide-binding sites. Some of the free Cys residues are surrounded by Phe aromatic rings, and mercury atoms come into contact with these residues in the derivative structure. It is strongly suggested that sulfur atoms bound to these sites could be stabilized by aromatic-electrostatic interactions by the surrounding Phe residues.

Fig. 1.

Oligomeric structure of Oligobrachia Hb. (A) Space-filling models of Oligobrachia Hb 24-mer viewed along with the crystallographic 3-fold axis (Upper) and 2-fold axis (Lower). Four individual globin subunits A1, A2, B1, and B2 are shown in red, green, yellow, and blue, respectively. (B) Stereo view of the Hb tetramer composed of one copy of each of four globin subunits colored as in A. Each helix is labeled according to the myoglobin helical designations. There is no D helix in any of the four globins and no C helix in B1. All four globin subunits form intramolecular disulfide bonds between their N terminus and the middle of their H-helix; these bonds are shown as cyan balls. Intermolecular disulfide bonds are also shown as light yellow balls formed between the A1 GH corner and B2 GH corner, and as magenta balls formed between the B1 N terminus and B2 N terminus of the neighboring tetramer (not shown).

Fig. 2.

The σ_A-weighted 2F_o - F_c map contoured at the 1σ level around the B2 heme site shown as a cyan mesh. The F_o - F_c map calculated with a ligand-omitting model is also shown in magenta, contoured at the 3σ level. In the 2F_o - F_c map, the electron density of the second oxygen atom of O₂ is not clear, probably because of different orientations in the crystal. The position of the oxygen atom bonded to the Fe of heme was restrained during refinements.

Fig. 3.

Interactions of the EF dimer interfaces. (A) Oligobrachia A1B1 dimer. (B) A2B2 dimer. (C) Lumbricus A1B1 dimer. E and F helices are represented as Cα traces, and the key residues coming into contact with the neighboring heme propionate group are drawn as sticks together with heme molecules. The Cα traces and heme molecules of Oligobrachia A1, A2, B1, and B2 and Lumbricus A1 and B1 are red, green, yellow, blue, and gold, respectively. The interactions of Lumbricus A2B2 dimer and Riftia A1B1 and A2B2 dimer interfaces appear almost the same manner as that of Lumbricus A1B1 dimer. Only in the case of Oligobrachia A2, the position F3 is Arg and forms a hydrogen bond with the other propionate group of which His-F3 of A1, B1, and B2 forms a hydrogen bond.

Fig. 4.

The environments around free cysteine residues at the proposed sulfide-binding sites. The positions of the mercury atoms in the Hg derivative structure are represented as magenta balls, indicating the putative sulfide positions. (A) The conserved free Cys site (E7+1) of the A1 surrounded by Phe aromatic rings. Because the Cys residue is in a subsequent position to distal His, this site is located almost right above the heme distal pocket, with the atomic distance between the Fe of the heme and the S of the Cys being ≈10 Å. (B) Another conserved free Cys site (E7+11) of the A2. No striking interactions with the putative sulfide position were observed. (C) B1 Cys-E7+21 is not a conserved residue, but Tyr-136 is located at a suitable position for stabilizing bonded sulfide. (D) The E7+1 site of the B2, which is quite similar to the A1 shown in A.