Cytochrome c : Folding Triggered by Electron Transfer. Rates of Heme Oxidation and Reduction at High Driving Forces

Citation

Mines, Gary Alan (1997) Cytochrome c : Folding Triggered by Electron Transfer. Rates of Heme Oxidation and Reduction at High Driving Forces. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rtx8-jn96. https://resolver.caltech.edu/CaltechTHESIS:07152025-200013414

Abstract

Rates of various intramolecular heme oxidations and reductions in a series of closely related RuL₂(X)(His33)cytochromes c [L = bipyridine or phenanthroline derivatives; X = imidazole (im) or cyanide (CN^-)] have been measured over a freeenergy range of 0.54 to 1.89 eV. The driving-force dependence of Fe²⁺→Ru³⁺ electron transfer (ET) is well described by semiclassical ET theory with a coupling-limited rate (k_max) of 2.8 x 10⁶ s^-1 and a reorganization energy of 0.74 eV. As predicted by theory, the rate of an exergonic (-ΔG° = 1.3 eV) heme reduction reaction, *Ru²⁺(bpy)₂(im)(His)→Fe³⁺, falls in the inverted region (k = 2.0 x 10⁵ s^-1). In contrast, the rates of three highly exergonic heme reductions, *Ru²⁺(phen)₂(CN)(His)→Fe³⁺ (3.1 x 10⁵ s^-1; 1.4 eV), Ru⁺(4,4'-(CONH(C₂H₅))₂-bpy)₂(im)(His)→Fe³⁺ (2.3 x 10⁵ s^-1; 1.44 eV), and Ru⁺(phen)₂(CN)(His)→Fe³⁺ (4.5 x 10⁵ s^-1; 1.89 eV), are much higher than expected for reactions directly to ground-state products. Agreement with theory is greatly improved by assuming that an electronically excited ferroheme (Fe²⁺→*Fe²⁺ ~ 1.05 eV) is the initial product in each of these reactions.

In a separate investigation, rates of folding of ferrocytochromes c from horse (h-cyt c) and yeast (y-cyt c) were measured over a range of denaturant concentrations (guanidine hydrochloride, GuHCl) and folding free energies (ΔG_f) using a new ET triggering technique. The backbone structures of the two homologs are similar, but y-cyt c is ~ 15 kJ mol^-1 less stable than h-cyt c and is unfolded at concentrations of GuHCl ~ 1.5 M lower than for h-cyt c. Activation free energies exhibit a linear dependence on GuHCl and ΔG_f for both proteins, with folding rates decreasing with increasing concentration of GuHCl (less negative ΔG_f). At a given denaturant concentration, the folding rates for y-cyt c are about an order of magnitude slower than those for h-cyt c, but when the folding free energies are matched, folding rates of the two homologs are comparable.

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