Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

2006

Thesis Director

Scott Tremain

Thesis Committee Member

Jonathan Blitz

Thesis Committee Member

Gopal Periyannan

Thesis Committee Member

Kraig Wheeler

Abstract

We study, by absorbance spectroscopy, the incorporation of various metal ions (Cu²+ and Zn2*) into metal-free porphyrin cytochrome c (H₂cyt) as well as the various conformational forms of metal-substituted cytochromes. The H2cyt and desired metalsubstituted cytochromes have significantly different absorption spectra, providing a simple means of monitoring the incorporation of Cu²* or Zn2* into the empty porphyrin prosthetic group. The rates of metal-ion incorporation were studied under protein denaturating conditions using chemical denaturants, like urea and guanidine hydrochloride (GuHCI), and temperature that partially and completely unfold H2cyt, facilitating the incorporation of a metal ion. Moreover, the structural stability of copper substituted cytochrome c (Cucyt) was investigated using fluorescence spectroscopy.

In general, increasing concentrations of urea and GuHCI significantly affect the rate of metal-ion incorporation into H2cyt and also lead to the appearance of various structural conformations of metal-substituted cytochromes that can be resolved via absorbance spectroscopy. At lower concentrations of denaturant, the rate of incorporation of a metal ion was slower due to less exposure of the buried porphyrin to solvent as cytochrome c is only partially unfolded. Moreover, metal-ion incorporation was incomplete, as some H₂cyt remained (confirmed by absorbance spectroscopy). However, at higher concentrations of denaturant, the rate of incorporation was rapid due to the highly solvent-accessible porphyrin in unfolded cytochrome c. No H2cyt was spectroscopically observed after incorporation, indicating complete incorporation of the desired metal ion. Moreover, the rate constants for the decay of H₂2cyt and the formation of metal-substituted cytochrome c are similar, indicating a single step process without any intermediates.

Specifically, the rate constant for the incorporation of Zn²* into H2cyt initially increases as GuHCI concentration increases; however, above 2.0 M GuHCI, the rate constants remain unchanged. This indicates that at 2.0 M GuHCI, the porphyrin is already maximally accessible for Zn2* incorporation. Overall, the rate of Cu2+ incorporation into H2cyt is faster than Zn²* incorporation. Upon incorporation of Cu²* into H2cyt,various spectroscopic forms of Cucyt were observed. The shift in the heme Soret absorbance band was dependent on the concentration of GuHCl. At lower GuHCI concentrations (0.5 M & 1.0 M), two Soret absorbance bands at 403 nm and 422 nm were observed, indicating the presence of two different Cucyt structural conformations. At higher concentrations of GuHCI (2.5 M4.0 M), a single Soret absorbance band at 390 nm was observed, indicating a third Cucyt structural conformation. In drastic contrast to Cucyt, only one spectroscopic form of Zncyt has been observed under the same experimental conditions, with a Soret absorbance band at 423 nm. These different spectroscopic forms of Cucyt are most likely due to differences in the coordination environment ofthe Cu2* ion upon changes in axial ligation.

In one experimental preparation of H2cyt, incorporation of an unknown contaminating metal ion was observed prior to any addition of the desired metal ion salt solution to the cuvette solution. Prompted by this experimental observation, we wanted to determine the lowest concentration of GuHCl that still allowed contaminating metal ion incorporation. Metal-ion incorporation can be a sensitive probe of the protein's tertiary structure and porphyrin accessibility. The goal was to incrementally lower the GuHCI concentration until no GuHCl-induced conformational changes in H2cyt were observed. Even at very low GuHCI concentrations (0.1 M, 0.01 M, and 0.001 M), metalion incorporation was observed indicating GuHCl-induced conformation changes (i.e. partial protein unfolding and increased porphyrin accessibility). At 0.0001 M GuHCI, no GuHCl-induced conformational changes were observed.

To determine the structural stability of Cucyt, fluorescence spectroscopy was used to monitor the decrease in tryptophan fluorescence emission intensity as denaturant concentration increases. Urea-induced and GuHCI-induced equilibrium unfolding studies of Cucyt indicated a midpoint for the unfolding transition (C) in urea at 4.7±0.1 M and in GuHCI at 2.6±0.2 M. The extrapolated free energy for unfolding Cucyt in the absence of denaturant (AGU) was 19.4±5.5 kJ/mol using GuHCl as a denaturant and 23.8±2.3 kJ/mol using urea as a denaturant.

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