title: Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin.
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 O2, 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.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Knappenberger
authors.forename: Syna A
authors.initials: SA
authors.lastname: Kuriakose
authors.forename: B Christie
authors.initials: BC
authors.lastname: Vu
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Nothnagel
authors.forename: David A
authors.initials: DA
authors.lastname: Vuletich
authors.forename: Juliette T J
authors.initials: JT
authors.lastname: Lecomte
mesh_terms: D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003486:Cyanides / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005914:Globins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D050661:Sperm Whale; D013329:Structure-Activity Relationship; D046939:Synechocystis / Q000737:chemistry*; D013696:Temperature; D014508:Urea / Q000378:metabolism
chemical_list: D003486:Cyanides; D005290:Ferric Compounds; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D012694:Serine; D006639:Histidine; D014508:Urea; D005914:Globins; D000409:Alanine