Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands.
Three hemoglobins have been isolated from the symbiont-harboring gill of the bivalve mollusc Lucina pectinata. Oxyhemoglobin I (Hb I), which may be called sulfide-reactive hemoglobin, reacts with hydrogen sulfide to form ferric hemoglobin sulfide in a reaction that may proceed by nucleophilic displacement of bound superoxide anion by hydrosulfide anion. Hemoglobins II and II, called oxygen-reactive hemoglobins, remain oxygenated in the presence of hydrogen sulfide. Hemoglobin I is monomeric; Hb II and Hb III self-associate in a concentration-dependent manner and form a tetramer when mixed. Oxygen binding is not cooperative. Oxygen affinities are all nearly the same, P50 = 0.1 to 0.2 Torr, and are independent of pH. Combination of Hb I with oxygen is fast; k'on = (estimated) 100-200 x 10(6) M-1 s-1. Combination of Hb II and Hb III with oxygen is slow: k'on = 0.4 and 0.3 x 10(6) M-1 s-1, respectively. Dissociation of oxygen from Hb I is fast relative to myoglobin: koff = 61 s-1. Dissociation from Hb II and Hb III is slow: koff = 0.11 and 0.08 s-1, respectively. These large differences in rates of reaction together with differences in the reactions of carbon monoxide suggest differences in configuration of the distal heme pocket. The fast reactions of Hb I are comparable to those of hemoglobins that lack distal histidine residues. Slow dissociation of oxygen from Hb II and Hb III suggest that a distal residue may interact strongly with the bound ligand. We infer that Hb I may facilitate delivery of hydrogen sulfide to the chemoautotrophic bacterial symbiont and Hb II and Hb III may facilitate delivery of oxygen. The midpoint oxidation-reduction potential of the ferrous/ferric couple of Hb I, 103 +/- 8 mV, was independent of pH. Potentials of Hb II and Hb III were pH-dependent. At neutral pH all three hemoglobins have similar midpoint potentials. The rate constant for combination of ferric Hb I with hydrogen sulfide increases 3000-fold from pH 10.5 to 5.5, with apparent pK 7.0, suggesting that undissociated hydrogen sulfide is the attacking ligand. At the acid limit combination of ferric Hb I with hydrogen sulfide, k'on = 2.3 x 10(5) M-1 s-1, is 40-fold faster than combination with ferric Hb II or myoglobin.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
D W
DW
Kraus
J B
JB
Wittenberg
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D049872:Bivalvia / Q000378:metabolism*; D002263:Carboxyhemoglobin / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010108:Oxyhemoglobins / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D013440:Sulfides / Q000378:metabolism; D013559:Symbiosis
D000596:Amino Acids; D006454:Hemoglobins; D008024:Ligands; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D013440:Sulfides; D006418:Heme; D002263:Carboxyhemoglobin