Building reactive copper centers in human carbonic anhydrase II.

Song, He; Weitz, Andrew; Hendrich, Michael; Lewis, Edwin; Emerson, Joseph

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 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-binding events have different affinities ( K approximately 5 × 10 and 1 × 10), and both are enthalpically driven processes. Reconstituting these Cu sites under a range of conditions has allowed us to assign the Cu-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu 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.

COPPER; CARBONIC anhydrase; METALLOPROTEINS; PROTEIN engineering; THERMODYNAMICS; BINDING sites; ISOTHERMAL titration calorimetry

Journal of Biological Inorganic Chemistry (JBIC), 2013, Vol 18, Issue 6, p595

0949-8257

Academic Journal

10.1007/s00775-013-1009-1