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Electron transfer properties of flavin-modified and axial ligand variants of cytochrome c

University of British Columbia

Electron transfer (ET) reactions of metalloproteins are central to many biological processes. This fact combined with the simplicity of the chemistry involved makes this type of reaction amenable to detailed experimental and theoretical analysis. The current work employs two novel forms of mitochondrial cytochrome c to investigate critical structural factors that dictate the efficiency of intermolecular and intramolecular ET reactions of this protein and that have implications for metalloproteins in general. The first of these approaches involved the use of a new technique to study intramolecular ET reactions in which a combination of site-directed mutagenesis and chemical modification was used to construct a family of synthetic flavocytochromes c. Previous studies by Tollin and Cusanovich have used flavins extensively to investigate intermolecular ET reactions of electron transfer proteins, but flavins have not been used previously as donors in the study of intramolecular ET reactions. In the present work, flash photolysis was used to study the intramolecular ET kinetics of four cytochrome variants bearing a flavin group at position 8, 39, 62, or 85. To complement this work, the theoretical models of Beratan, Betts and Onuchic and of Siddarth and Marcus were used to identify the theoretically optimal ET pathways and donor-acceptor electronic coupling for each of these derivatives. A major finding derived from the kinetic studies of these synthetic flavocytochromes is that the reorganization energy for electron transfer, λ, of the flavin center is 0.7(2) eV. In addition, the theoretical models for these reactions demonstrate that the correlation of the rate constants for intramoleculer ET observed can be understood in terms of the nature and length of the ET pathway between the donor and acceptor centers. The second approach described in this thesis involved the study of three cytochrome c variants in which the Met80 ligand to the heme iron was replaced with an alanyl residue. Analysis of the intermolecular reduction of these variants by Fe(EDTA)²⁻ was undertaken to gain insight into the role of the Met80 ligand and two other active site residues in regulating the electron transfer reactivity of the cytochrome. These variants exhibited a remarkable range in reactivity with this reductant as represented by the apparent electrostatics-corrected self-exchange rate constants, k₁₁ [sup corr], exhibited in this reaction (pH 7, μ= 0.1 M, 25 °C): Met80Ala, k₁₁[sup corr] = 1.1 M⁻¹ s⁻¹; Met80Ala/Tyr67Phe, k₁₁[sup corr] = 3.4 x 102; and Met80Ala/Phe82Ser, k₁₁ [sup corr]= 4.3 x lO⁻² M ⁻¹s⁻¹. Possible explanations of these results are discussed.

Thesis/Dissertation

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2009-05-28

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http://hdl.handle.net/2429/8437

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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