title: Building reactive copper centers in human carbonic anhydrase II.
abstract: Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu(2+) ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu(2+)-binding events have different affinities (K a approximately 5 × 10(12) and 1 × 10(10)), and both are enthalpically driven processes. Reconstituting these Cu(2+) sites under a range of conditions has allowed us to assign the Cu(2+)-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu(2+) sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.
authors.affiliation: Department of Chemistry, 1115 Hand Lab, Mississippi State University, Mississippi State, MS 39762-9573, USA.
authors.forename: He
authors.initials: H
authors.lastname: Song
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Weitz
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hendrich
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Lewis
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Emerson
mesh_terms: D000627:Aminophenols / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D013816:Thermodynamics
chemical_list: D000627:Aminophenols; D009942:Organometallic Compounds; C027667:2-aminophenol; D003300:Copper; D006861:Hydrogen Peroxide; D024402:Carbonic Anhydrase II