Mechanistic Investigations into Cytochrome c and Water-Soluble Porphyrin Model Systems

Abstract

Heme-dependent enzymes represent a diverse class of proteins capable of mediating redox transformations. Due to their importance in the regulation of cell health, considerable research has been performed on both enzymatic systems and porphyrin model systems. This work investigates the mechanism of previously uncharacterized reactivity in both enzymatic and model systems.

In chapter 2, we investigate the previously unidentified nature of the heme enzyme, cytochrome c (cyt c), as a peroxygenase. Using mass spectrometry (MS), we confirm previous reports of cyt c ability to transfer the acyl tail of fatty-acyl coenzyme A-moieties to primary amines. Additionally, we observe the incorporation of labeled 18O from H218O2 into unsaturated acyl tails. Using MS, NMR, and MS/MS, we demonstrate this oxygen incorporation results in a cis-epoxide product. Moreover, sulfenic acid traps are utilized to reveal an introductory sulfoxidation step activates the CoA-moiety for acyl transfer. Each of these oxidations represents a 2e- process in which oxygen is transferred from the peroxide into the product. The biological implications of this newfound peroxygenase activity are discussed.

In chapter 3, we investigate the reactivity of Mn-substituted porphyrin model systems and compare their reactivity with the corresponding iron porphyrins. The current sequential two-proton oxidation model is compared to single-proton and inverted pKa models for water-soluble Mn porphyrins. Additionally, Eyring analysis is used to determine the thermodynamic activation parameters in C-H abstractions. These studies suggest that the metal-oxo pKa mediates the thermodynamic driving force of the reaction.

Finally, in chapter 4, the oxidation of formate by water-soluble Mn porphyrins is studied. A combination of stopped-flow spectroscopy and electrochemistry is used to demonstrate that oxidation of formate proceeds rapidly in a pH-dependent manner (~101-106M-1s-1), far outpacing the current standard in metal-oxo catalysts. Deuterium kinetic isotope effects (KIE) and solvent KIE are used in combination with substrate analog studies to interrogate the mechanism of this reaction. This work results in the proposal of a decarboxylation-coupled hydride transfer pathway resulting in the 2e- reduction of MnV to MnIII.