SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata.

Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.

Two-step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4-9.

Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.

Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complex.

The native oxygen-carrier haemoglobins complex (HbII-III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20 A, respectively, using a 0.886 A synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P4(2)2(1)2, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49 A for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (V(M)) of 3.08 and 3.06 A(3) Da(-1) for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume.