Haemoglobin structure and biochemical characteristics of the sulphide-binding component from the deep-sea clam Calyptogena magnifica

Calyptogena magnifica is a large heterodont clam belonging to the family ve sicomyidae that lives at a depth of ca. 2600 m, near deep-sea hydrothermal vent areas on the East-Pacific Rise and Galapagos Rift. This species harbor s abundant autotrophic sulphide-oxidizing bacteria contained in bacteriocyt es and located in the gills. Unlike the tube-worm Riftia pachyptila, which possesses extracellular haemoglobins that bind sulphide and oxygen simultan eously and reversibly at two different sites, Calytogena magnifica possesse s in its haemolymph two different types of molecules for these functions: a n intracellular circulating haemoglobin (Hb) for oxygen binding and a sulph ide-binding component (SBC) dissolved in the serum. To elucidate its quater nary structure, the haemoglobin was purified by gel chromatography (FPLC) a nd then analysed by electrospray ionization mass spectrometry (ESI-MS). FPL C analysis revealed a molecular mass of approximately 68 kDa. By mass spect rometry, under native condition, we found that this molecule contains two s ubunits of molecular masses 16134.0 Da (alpha) and 32513.1 Da (beta gamma). After reduction, the py subunit corresponds to a covalent heterodimer cons isting of chains beta and gamma with Mr of 16148.0 and 16371.0, respectivel y. Our data suggest that C. magnifica intracellular Hb is a tetrameric mole cule with three possible associations: (beta gamma)(2) (beta gamma)(1) (alp ha)(2), and/or (alpha)(4). The electron micrographs show that the sulphide- binding serum component is a dumbbell-shaped molecule consisting of two glo bular subunits of 74 + 5 nm. It is a glycosylated molecule (non-haeme) that also possesses a protein moiety, Its absorbance peak shifts from 280 nm to 208 nm when it binds sulphide. This molecule moreover possesses unusual so lubility properties suggesting that it may be a lipoprotein. It has a high zinc content and there is a 1:1 ratio between zinc and sulphide bound sugge sting that zinc is the sulphide-binding site of this compound.