Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus.

A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 Angstroms resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric.

[1]  D. McRee.  Practical Protein Crystallography , 1993 .

[2]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[3]  Veronica Morea,et al.  The Truncated Oxygen-avid Hemoglobin from Bacillus subtilis , 2005, Journal of Biological Chemistry.

[4]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[5]  Veronica Morea,et al.  A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca , 2005, The FEBS journal.

[6]  K. Wilson,et al.  Efficient anisotropic refinement of macromolecular structures using FFT. , 1999, Acta crystallographica. Section D, Biological crystallography.

[7]  M. Guertin,et al.  Unique ligand-protein interactions in a new truncated hemoglobin from Mycobacterium tuberculosis. , 2002, Biochemistry.

[8]  Paolo Ascenzi,et al.  A TyrCD1/TrpG8 hydrogen bond network and a TyrB10—TyrCD1 covalent link shape the heme distal site of Mycobacterium tuberculosis hemoglobin O , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  David Dantsker,et al.  Reactions of Mycobacterium tuberculosis truncated hemoglobin O with ligands reveal a novel ligand-inclusive hydrogen bond network. , 2003, Biochemistry.

[10]  A. Bhuyan,et al.  Open reading frame yjbI of Bacillus subtilis codes for truncated hemoglobin. , 2005, Protein expression and purification.

[11]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[12]  J. Hajdu,et al.  Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide. , 2005, Biochemistry.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  D. Rousseau,et al.  A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Martino Bolognesi,et al.  Truncated Hemoglobins: A New Family of Hemoglobins Widely Distributed in Bacteria, Unicellular Eukaryotes, and Plants* 210 , 2002, The Journal of Biological Chemistry.

[16]  Alessandra Pesce,et al.  Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins. , 2005, Journal of inorganic biochemistry.

[17]  S. Kundu,et al.  Direct measurement of equilibrium constants for high-affinity hemoglobins. , 2003, Biophysical journal.

[18]  D. Rousseau,et al.  Pentacoordinate hemin derivatives in sodium dodecyl sulfate micelles: model systems for the assignment of the fifth ligand in ferric heme proteins. , 1999, Biophysical journal.