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Structural-functional characterization of the cathodic haemoglobin of the conger eel Conger conger: molecular
Mariagiuseppina Pellegrini1, Bruno Giardina, Cinzia Verde
1Department of Sciences Applied to Biosystems, University of Cagliari, Cittadella Universitaria, I-09042 Monserrato (CA), Italy. pelleg@unica.it
Insights
Conger eel cathodic hemoglobin (Hb) exhibits a reverse Bohr effect. Guanine triphosphate (GTP) binding to two sites modulates this effect, influencing oxygen affinity through specific amino acid interactions.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Chemistry
Background:
- The Conger conger (conger eel) hemoglobin (Hb) system comprises three components.
- The cathodic Hb, approximately 20% of the total pigment, was purified and characterized.
Purpose of the Study:
- To structurally and functionally characterize the conger eel cathodic Hb.
- To investigate the effect of guanine triphosphate (GTP) on Hb oxygen affinity and Bohr effect.
- To elucidate the molecular mechanisms of GTP binding and its influence on Hb function.
Main Methods:
- Purification and characterization of cathodic Hb.
- Oxygen binding measurements under various conditions (stripped, with GTP).
- Computer modeling based on determined alpha- and beta-chain amino acid sequences.
Main Results:
- Stripped cathodic Hb displayed a reverse Bohr effect, high oxygen affinity, and low cooperativity.
- Saturating GTP converted the reverse Bohr effect to a small normal Bohr effect.
- GTP titration indicated two independent binding sites, likely involving hydrogen bonds and electrostatic interactions with identified residues, including Lys-alpha(G6).
Conclusions:
- Conger eel cathodic Hb binds organic phosphates, like GTP, at two distinct sites within the central tetramer cavity.
- Specific amino acid residues, notably Lys-alpha(G6), play a crucial role in GTP binding and allosteric modulation of Hb function.
- The findings provide insights into the structural basis of oxygen transport regulation in fish hemoglobin.
Abstract:
The protein sequence data for the alpha- and beta-chains have been deposited in the SWISS-PROT and TrEMBL protein knowledgebase under the accession numbers P83479 and P83478 respectively. The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-alpha(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.