Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes

Abstract

Visible circular dichroism, absorption, and fluorescence spectroscopy were used to probe the binding of horse heart ferrocytochrome c to anionic cardiolipin (CL) head groups on the surface of 1,1',2,2'-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in an aerobic environment. We found that ferrocytochrome c undergoes a conformational transition upon binding that leads to complete oxidation of the protein at intermediate and high CL concentrations. At low lipid concentrations, the protein maintains a structure that is only slightly different from its native one, whereas an ensemble of misligated predominantly hexacoordinated low-spin states become increasingly populated at high lipid concentrations. A minor fraction of conformations with either high- or quantum-mixed-spin states were detected at a CL to protein ratio of 200 (the largest one investigated). The population of the non-native state is less pronounced than that found for cytochrome c-CL interactions initiated with oxidized cytochrome c. Under anaerobic conditions, the protein maintains its reduced state but still undergoes some conformational change upon binding to CL head groups on the liposome surface. Our data suggest that CL-containing liposomes function as catalysts by reducing the activation barrier for a Fe → O electron transfer. Adding NaCl to the existing cytochrome-liposome mixtures under aerobic conditions inhibits protein autoxidation of ferrocytochrome c and stabilizes the reduced state of the membrane-bound protein.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

“…ITC experiments were performed under aerobiosis to mimic physiological conditions, despite using degassed samples. This may result in partial oxidation of Cc upon binding to liposomes, as previously reported (71). ITC analysis yielded the apparent K D values for the first binding event of 427 μM (WT Cc) and 780 μM (Y48pCMF Cc) (Fig.…”

Relating the multi-functionality of cytochrome c to membrane binding and structural conversion

“…The authors inferred an equilibrium constant of 1.4•10 4 M −1 from their data, but they did not elaborate on the utilized algorithm used for their analysis. Apparently, this value is even lower than the K 2 constant reported by Sinibaldi et al (2008) Several studies exploring the binding of CL-containing SUVs have been conducted in our own laboratory over the last 3 years Schweitzer-Stenner 2014, 2015a, b;Serpas et al 2016). Figure 15 exhibits different sets of spectroscopic responses to the binding of cyt o to TOCL/DOPC SUVs with different TOCL content (20, 50, and 100%) at neutral pH Schweitzer-Stenner assumed two different binding sites on the protein (Pandiscia and Schweitzer-Stenner 2015a).…”

Polyethylene glycol promotes autoxidation of cytochrome c

“…Explanation for the rapid autoxidation of heme iron in the presence of PEGIn this paper, we found that the heme iron of Cyt c was rapidly oxidized in the presence of 5-20% PEG(Figure 1). The oxidation rate of reduced Cyt c was recently reported to increase through interactions with the mitochondrial lipid, cardiolipin (CL)[43,44]. The absorption spectra of reduced Cyt c in the presence of 200 M CL exhibited a blue shift in the Soret maximum, from 413.5 to 408.5 nm, which suggested that CL induced a conversion to a non-native state[44][45][46].…”

“…In this case, the absorbance at 695 nm decreased as the CL concentration increased. Additionally, the CD signal was altered from a couplet to a positive Cotton effect band, reflecting a transition from a native state to a partially unfolded state[44,46,47]. Especially, Bradley reported that in the reduced Cyt c-CL complex, the major conformer (80%) contains high-spin heme iron, suggesting that heme is predominantly five-coordinate, but not six-coordinate, in the presence of CL and thus readily binds small exogenous ligands such as O2[48].…”