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Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin
- Source :
- Biochemistry. 45:11401-11413
- Publication Year :
- 2006
- Publisher :
- American Chemical Society (ACS), 2006.
-
Abstract
- The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O 2 , CO, and CN - , among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala → Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.
- Subjects :
- Protein Denaturation
Heme binding
Molecular Sequence Data
Heme
Ferric Compounds
Biochemistry
Article
Structure-Activity Relationship
chemistry.chemical_compound
Serine
Urea
Histidine
Denaturation (biochemistry)
Amino Acid Sequence
Globin
Leghemoglobin
Nuclear Magnetic Resonance, Biomolecular
Alanine
Cyanides
Sperm Whale
biology
Myoglobin
Synechocystis
Temperature
Hexacoordinate
Hydrogen-Ion Concentration
biology.organism_classification
Recombinant Proteins
Globins
Kinetics
Amino Acid Substitution
chemistry
Protein Binding
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 45
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....0ba8da8344deccd2f0db4d49137a9d9f