Spectroelectrochemical Studies of Heme and Copper Proteins

Citation

Ellis, Walter Robert, Jr. (1986) Spectroelectrochemical Studies of Heme and Copper Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fqg8-e811. https://resolver.caltech.edu/CaltechTHESIS:08062025-201831641

Abstract

A complete characterization of the redox thermodynamics of a metalloprotein involves the determination of the effects of pH, ionic strength, and temperature. In particular, variable-temperature reduction potential measurements yield the enthalpic and entropic contributions to E^O, which, in turn, are related to kinetic activation parameters.

In Chapter II of this thesis, the design of a versatile spectroelectrochemical apparatus for high precision measurements of metalloprotein reduction potentials is described. A stainless steel shroud, necessary for anaerobic measurements, is used to house either of two types of spectroelectrochemical cells: a thin-layer optically transparent electrode cell, or a two cm. stirred cell (useful for observing weak chromophores). Both types of cells are suitable for variable-temperature measurements using either absorption or circular dichroism spectroscopy. The latter offers a useful alternative to absorption spectroscopy since redox mediators, which are almost always necessary for electrochemical experiments on metalloproteins, are generally not optically active and therefore will not interfere with the acquisition of protein circular dichroism spectra.

Spectroelectrochemical measurements for a selection of paradigmatic hemoproteins are presented in Chapter III. Horse heart cytochrome c is shown to yield redox thermodynamic parameters in excellent agreement with results of calorimetric determinations. Data for R. palustris cytochrome c' and myoglobin are also presented and discussed with reference to extant spectroscopic and kinetic data, and crystal structure determinations for these proteins. ΔS^O does not correlate with protein surface charge, unlike the well-known correlation of ΔS^O with charge for transition metal complexes. Instead, it is suggested that ΔS^O is (at least in part) determined by redox-linked protein conformational changes. Next, redox thermodynamic parameters for the azurins from A. denitrificans and A. faecalis are presented. The azurin data show that proton uptake (in the region pK_a^ox less than pH less than pK_a^red) accompanying the reduction of the copper site decreases ΔS^O slightly. This observation is discussed in light of crystallographic studies on "blue" copper proteins.

Chapter IV discusses the problems of temperature and metal-metal interactions associated with the equilibrium redox behavior of multisite metalloproteins. An analogy between allosteric ligand binding and allosteric electron binding (i.e., reduction) is used to analyze Nernst plots exhibiting unusual slopes. In interactive situations, the conventionally defined reduction potential is not a simple thermodynamic quantity. This formalism is used in a discussion of the equilibrium redox interactions displayed by the optically visible metal centers (Cu_A, Fe a, Fe a₃) in cytochrome c oxidase. The commonly accepted "neoclassical" model, which only incorporates interaction between Fe a and Fe ₃, is shown to be incorrect. The importance of temperature (leading to varying entropies of reduction for different metal sites in a protein) is illustrated by a (NH₃)₅Ru(His-48)-derivative of sperm whale myoglobin. Kinetic and thermodynamic data for this two-site protein lead to an estimate of 20 kcal/mol for the reorganizational enthalpy of the iron heme in myoglobin. This value substantially larger than the 7-8 kcal/mol estimate for horse cytochrome c, and is most likely due to a ligation change upon reduction of myoglobin wherein the axial water molecule dissociates from the iron center.

A detailed discussion of the pH, ionic strength, and temperature dependences of the formal reduction potentials of the Cu_A, Fe a. and Fe a₃ centers in cytochrome oxidase is the topic of Chapter V. These results are compared with values for other protein copper and heme sites and are discussed within the context of the known structural features and reactivity of the enzyme.

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