The reduction potential (ε°) of a metal bound to a protein is determined by its coordination ligand field and environment, which can be partitioned into enthalpic (△He°) and entropic (△Se°) contributions to the free energy of reduction (△Ge° = n Fε°). However, these thermodynamic values for the reduction of a metalloprotein are not easily determined. A method to do so, which we call Redox Coordination Thermodynamics (RCT), involves quantifying the thermodynamics associated with both the oxidized metal and the reduced metal binding to the protein and analysis with thermodynamic cycles (see Figure). We have evaluated this method by isothermal titration calorimetry (ITC) measurements of Cu(II) and Cu(I) binding to azurin for comparison to the azurin reduction thermodynamics determined previously with temperature-dependent electrochemical methods. In addition to quantifying the reduction enthalpy and entropy, ITC measurements can also determine the (de)protonation that is coupled with each metal ion binding to the protein and, by the difference, quantify the (de)protonation that is coupled with reduction of the metalloprotein. The RCT method is now being used to quantify the thermodynamics that modulate the reduction potential of a series of azurin mutants prepared by Yi Lu and co-workers.

Thermodynamic contributions to the metalloprotein reduction potential

251st National Meeting (2016)

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