title: Manipulating cofactor binding thermodynamics in an artificial oxygen transport protein.
abstract: We report the mutational analysis of an artificial oxygen transport protein, HP7, which operates via a mechanism akin to that of human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of 13. Paradoxically, it also decreases heme binding affinity by a factor of 5 in the reduced state and 60 in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates for that. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins.
authors: Department of Physics, The City College of New York, New York, New York 10031, United States.
authors: Lei
authors: L
authors: Zhang
authors: J L Ross
authors: JL
authors: Anderson
authors: Ismail
authors: I
authors: Ahmed
authors: Jessica A
authors: JA
authors: Norman
authors: Christopher
authors: C
authors: Negron
authors: Andrew C
authors: AC
authors: Mutter
authors: P Leslie
authors: PL
authors: Dutton
authors: Ronald L
authors: RL
authors: Koder
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D005813:Genes, Synthetic; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006420:Hemeproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D013816:Thermodynamics
chemical_list: D006420:Hemeproteins; D006418:Heme; D010100:Oxygen