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
