J. Mol. Biol. (1996) 258 1–5 COMMUNICATION Structural Bases for Sulfide Recognition in Lucina pectinata Hemoglobin I Menico Rizzi 1 , Jonathan B. Wittenberg 2 , Alessandro Coda 1 Paolo Ascenzi 3 and Martino Bolognesi 1,4 * The X-ray crystal structure of the sulfide derivative of ferric Lucina pectinata 1 Dipartimento di Genetica e hemoglobin component I (HbI) has been determined at 1.9 Å resolution Microbiologia, Universita’ di (R-factor 0.186). The heme pocket structural organization of HbI is in Pavia, Via Abbiategrasso 207 keeping with its ligand binding properties. The fast sulfide association rate constant can be related to the presence of Gln(64)E7, as the heme distal 27100 Pavia, Italy residue, together with the protein structural properties in the CD-E distal 2 Department of Physiology region. Moreover, the very high sulfide affinity for HbI is reflected by and Biophysics, Albert the exceptionally slow ligand dissociation rate. The stabilization of the Einstein College of Medicine heme-bound sulfide molecule is achieved through hydrogen bonding to Bronx, NY 10461, USA Gln(64)E7, as well as by finely tuned aromatic-electrostatic interactions with the clustered residues Phe(29)B10, Phe(43)CD1 and Phe(68)E11. Such 3 Dipartimento di Biologia, a peculiar arrangement of phenylalanyl residues at the distal ligand Terza Universita’ di Roma binding site has not been observed before in the globin family, and is Via Ostiense 173, 00154 unique to HbI, a protein functionally devoted to sulfide transport. Roma, Italy  1996 Academic Press Limited 4 IST, Centro Biotecnologie Avanzate and Dipartimento di Fisica, Universita’ di Genova, Viale Benedetto XV 10, 16132 Genova, Italy Keywords: heme protein; monomeric mollusc hemoglobin; sulfide carrier; *Corresponding author crystal structure Hemoglobins from symbiont-harboring molluscs The symbiosis between molluscs and intracellu- lar chemoautotrophic bacteria is always character- ized by the presence of mollusc cytoplasmic Hbs, most probably located in the cytoplasm of the bacteriocyte gill cells, which are specialized to house symbionts fixing carbon dioxide into hexoses. The cytoplasmic Hbs transport oxygen and hydro- gen sulfide from the sea water to the bacterial symbiont, permitting the bacteria to sustain rapid hexose synthesis, and supply the host animal with its entire carbohydrate nutrition (Arp, 1991; Wittenberg & Kraus, 1991). Three Hb components have been isolated from the modified gill of the bivalve mollusc Lucina pectinata , living in the coastal sedimental area of Puerto Rico. Components HbII and HbIII, isolated as homodimers or heterotetramers of two similar (approximately 17 kDa) polypeptide chains, bind O 2 non-cooperatively with an affinity (K = 3.5 × 10 6 M -1 ) similar to that observed for sperm whale Mb (K = 1.2 × 10 6 M -1 ), and are responsible for its transport. The monomeric HbI component, on the other hand, binds O 2 with comparable affinity (K = 1.6 × 10 6 M -1 ), but is readily oxidized to the ferric HbI:sulfide complex in the presence of trace oxygen and hydrogen sulfide concentrations (see Kraus & Wittenberg, 1990; Kraus et al ., 1990). The affinity of the sulfide ligand for HbI is exceptionally high (K = 2.9 × 10 8 M -1 ), being about 5000-fold higher than that observed for the HbII and HbIII components, and for sperm whale Mb. Such a high affinity is achieved through fast association (k on = 6.0 × 10 4 M -1 s -1 ) and very slow dissociation (k off = 7.8 × 10 -4 s -1 ) processes (see Kraus & Witten- Abbreviations used: Hb, hemoglobin; Mb, myoglobin; r.m.s., root-mean-square. Amino acid residues have been identified by their three-letter code, sequence number (in parentheses), and by their topological position in the globin fold. Water molecules have been identified by the W prefix (see Rizzi et al. 1994). 0022–2836/96/160001–05 $18.00/0  1996 Academic Press Limited