Doctor of Philosophy
The peroxidase activity of the mitochondrial protein cytochrome c (cyt c) plays a critical role in triggering programmed cell death, or apoptosis. However, the native structure of cyt c should render this activity impossible due to the lack of open iron coordination sites at its heme cofactor. Despite its key biological importance, the molecular mechanisms underlying this structure-function mismatch remain enigmatic. The work detailed in this dissertation fills this knowledge gap by using mass spectrometry (MS) to decipher the central role that protein oxidative modifications and their associated structural changes play in activating the peroxidase function of cyt c.
Chapter 2 uses a suite of MS-based experiments to identify and characterize oxidative modifications in cyt c caused by the oxidant and canonical peroxidase substrate, H2O2. In doing so, we unravel the critical role that these in situ structural changes play in triggering the peroxidase activity of the protein via alteration of the coordination environment. Serendipitously, we also discover that certain functionally important oxidative modifications, particularly on Lys, can elude detection when using conventional bottom-up MS approaches. However, by applying top-down MS we could successfully detect these modifications.
Chapter 3 re-examines a popular and purportedly well-characterized model system for peroxidase-activated cyt c: cyt c treated with chloramine-T. By combining top-down MS with sample fractionation techniques, we uncover that this model system is in fact comprised of a broad ensemble of structurally and functionally distinct species. These species can be differentiated by the extent of oxidation at key Lys residues, which previously went undetected.
Chapter 4 expands on the previous chapters by probing the causal factors underpinning the production of oxidative modification products at Lys and other residues. We discover that Lys oxidation is catalyzed by the endogenous heme cofactor, while other transformations (e.g. Met oxidation) proceed via direct interaction with the oxidant.
Chapter 5 utilizes oxidized cyt c as a model system to test the compatibility of protein stability measurements in the gas phase to their counterparts in solution. Unlike many other protein systems, we discover that oxidized cyt c shows opposing stability trends in solution and in the gas phase.
Summary for Lay Audience
Proteins are large molecules comprised of amino acids that play important roles in many aspects of biology. Proteins adapt a variety of functions, depending on their structure. Cytochrome c (cyt c) is a protein found in the mitochondria of cells that normally functions as an electron transporter, which is possible because of the iron-containing heme group in cyt c. Cyt c also has an alternative function (as a peroxidase), which plays a key role in triggering programmed cell death, or apoptosis. Despite this importance, it is poorly understood how cyt c can have peroxidase function despite structural features that should render it inactive. In this dissertation, we use mass spectrometry (MS) to study how oxidants can interact with cyt c, altering its structure and accommodating its peroxidase function.
We first studied the effects of the oxidant hydrogen peroxide (H2O2) on the structure and function of cyt c (Chapter 2). We uncovered that cyt c is extensively modified by H2O2. Using MS, we determined that these oxidative modifications cause structural changes near the heme that enable peroxidase function. A key finding was that oxidation of one type of amino acid, lysine, was critical.
We next focused on another oxidant, chloramine-T (CT) (Chapter 3). CT-treated cyt c has long been a popular model system for studying apoptosis and is thought to be simple and well-characterized. Using a combination of MS and purification techniques, we discovered that CT-treated cyt c is actually a mixture of structurally and functionally distinct species. The main difference between these species was the presence of lysine oxidation at key positions on the protein.
We then explored the processes underlying the formation of oxidative modifications in cyt c (Chapter 4). We determined that the heme plays a key role in producing lysine oxidation, while other amino acids (e.g. methionine) were oxidized independently of the heme group.
Finally, we used oxidized cyt c to compare the differences in protein stability using MS versus conventional spectroscopic techniques in solution (Chapter 5). Unlike many other proteins, we found that oxidized cyt c showed opposing stability trends in these two types of measurements.
Yin, Victor, "Effects of Oxidative Modifications on the Structure and Non-Canonical Functions of Cytochrome c Studied by Mass Spectrometry" (2020). Electronic Thesis and Dissertation Repository. 7332.