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Thermodynamic studies of copper binding to azurin : development and application of a novel method to determine the reduction potential and reduction thermodynamics of metalloproteins / by Molly L. Croteau

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Author	Croteau, Molly L.
Title	Thermodynamic studies of copper binding to azurin : development and application of a novel method to determine the reduction potential and reduction thermodynamics of metalloproteins / by Molly L. Croteau
Date	2016
Abstract	Cupredoxins, such as stellacyanin, plastocyanin, amicyanin, rusticyanin, and azurin perform electron transfer (ET) reactions with partner protein(s) in the cell. The role of the redox cofactor in these proteins is played by a copper ion, which becomes oxidized or reduced in order to donate or receive one electron. These blue copper proteins share a similar copper coordination site with equatorial ligands consisting of two histidine imidazoles and one cysteine thiolate, as well as one or two weaker axial ligands, but their reduction potential (??) spans the range from +190 mV to +390 mV, with rusticyanin at an incredible +680 mV. It is still not well understood how Nature tunes these and other metalloprotein reduction potentials, when the cofactor and coordination site are similar. To provide insight into this tunability, the enthalpy (?H), entropy (?S), and free energy (?G?) of copper, both in its Cu2+ and Cu+ oxidation states, binding to apo-azurin have been determined by isothermal titration calorimetry (ITC). Cu2+ binding is enthalpically unfavorable and entropically favorable, which indicates that the coordination of Cu2+ to apo-azurin reorganizes hydrogen bonds and water molecules that are at the surface of the hydrophobic metal site, resulting in the favorable entropy. Cu+ binding is both enthalpically and entropically favorable, which is expected due to the entatic state of the metal site which is imposed by the protein structure that prefers Cu+. In comparison to one another, Cu2+ is more entropically favorable, but more enthalpically unfavorable than Cu+. Since the metal site of azurin prefers the +1 oxidation state, there is less solvent reorganization and greater favorable bond formation upon Cu+ coordination than Cu2+. A new method to determine the reduction potential and contributing reduction thermodynamics for metalloproteins has been developed and validated with azurin, and is called reduction coordination thermodynamics (RCT). The thermodynamic parameters for both Cu2+ and Cu+ binding to azurin, along with those for the reduction of aqueous copper were used in a thermodynamic cycle to quantify the reduction enthalpy (?H??), free energy (?G???), and subsequently the reduction entropy (?S??) and reduction potential (??). Others have determined these values with electrochemical methods, but these require a range of temperatures to obtain ?H?? and ?S? ?, and there are assumptions that are not accurate or ignored for aqueous proteins. The reduction thermodynamic values obtained for azurin with the RCT method corroborate with the literature values determined with electrochemical methods. The RCT method was then applied to three azurin variants with unknown ?H?? and ?S?? values. The variants consisted of mutations to the 1st coordination sphere, the 2nd coordination sphere, and a combination of both, and were: F114P, F114P/M121Q, and N47S/F114N/M121L. The resulting ?? values were comparable to directly-measured electrochemical ?? values, and ?H?? and ?S?? were reported for the first time along with the contributing ?H and ?S values for each Cu2+ and Cu+ binding.
Description	1 online resource (xvi, 229 leaves) : illustrations (some color)
Format	application/pdf
College	Dartmouth College -- Department of Chemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	Made available by the author under a Creative Commons Attribution-NonCommercial License.; Made available by the Trustees of Dartmouth College under a Creative Commons Attribution-NonCommercial License. https://creativecommons.org/licenses/by-nc/4.0/
DOI	https://doi.org/10.1349/ddlp.2161

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Thermodynamic studies of copper binding to azurin : development and application of a novel method to determine the reduction potential and reduction thermodynamics of metalloproteins / by Molly L. Croteau

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