Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity

Abstract

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H 2 O 2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role.

Delineating Heme-Mediated versus Direct Protein Oxidation in Peroxidase-Activated Cytochrome c by Top-Down Mass Spectrometry

“…27 The presence of LysCHO in CT-holo-cyt c and in other oxidationactivated forms of the protein had long been overlooked 27,28 but is now well accepted. 24,41 A key finding of our previous work 27 was that despite cyt c possessing 19 Lys residues that are equally solvent accessible, 60 only a small Lys cluster (53/ 55/72/73) was susceptible to LysCHO formation (Figure 1). The factors responsible for this unexpected specificity remain unexplored.…”

“…The former ruptures the Fe–Met80 bond and generates the vacant coordination site required for catalysis, while the latter ensures that this site remains vacant by preventing Fe–Lys coordination . The presence of LysCHO in CT-holo-cyt c and in other oxidation-activated forms of the protein had long been overlooked , but is now well accepted. , A key finding of our previous work was that despite cyt c possessing 19 Lys residues that are equally solvent accessible, only a small Lys cluster (53/55/72/73) was susceptible to LysCHO formation (Figure ). The factors responsible for this unexpected specificity remain unexplored.…”

“…In addition to oxidative modifications that occur within living cells, ROS can induce protein oxidation in vitro . Experimental strategies that use this approach for monitoring the solvent accessibility of individual side chains have been developed. − In vitro oxidative modifications also provide the opportunity to investigate oxidation-induced changes in protein structure, dynamics, and function. ,,,− …”

“…Oxidative modifications of cytochrome c [cyt c (Figure )] have attracted a great deal of attention over the past few years. ,,,,,,− Cyt c acts as an electron transporter in the respiratory chain by cycling between its Fe­(II) and Fe­(III) heme oxidation states . In addition, cyt c can act as a peroxidase that catalyzes the oxidation of cardiolipin in mitochondria, a key step during apoptosis. ,− Interestingly, the peroxidase activity of native cyt c is very low because the heme iron is fenced in by a six-coordinate environment, being ligated by four pyrrole nitrogens, His18, and Met80.…”

Chemical Penetration Enhancers Increase Hydrogen Peroxide Uptake in C. elegans for In Vivo Fast Photochemical Oxidation of Proteins

“…Fast photochemical oxidation of proteins (FPOP) is an HRPF method that utilizes a 248 nm KrF excimer laser for photolysis of hydrogen peroxide to label proteins on the microsecond time scale in the absence of secondary radicals. − In vitro applications of FPOP include the study of protein conformers, conformational changes, and protein–ligand interactions. − Recently, we expanded the use of FPOP for the study of protein structure in vivo in Caenorhabditis elegans (C. elegans), in a new method entitled in vivo FPOP (IV-FPOP) …”