Polyethylene glycol promotes autoxidation of cytochrome c

Abstract

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV-visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.

Interaction of polyethylene glycol with cytochrome c investigated via in vitro and in silico approaches

“…In addition, the study proposed a model showing that heme is dislocated due to dehydration of some surface exposed hydrophobic residues i.e. Ile81 and Val83; however their spectroscopic techniques showed no significant changes 31 and are comparable to our results at lower concentrations. They have used small concentrations of PEG 4 kDa, however we are dealing with macromolecular crowding and its effect on the protein we have used variable concentrations (low to high) of the same molecular weight of PEG in similar conditions but through different approaches.…”

“…1 A,B). Previously it has been reported that PEG 4 kDa of small concentrations has little change without any shift in Soret absorption and oxy-deoxy band in cyt c 31 . The studies claim that PEG 4 kDa promotes auto-oxidation of cyt c , however changes are observed to be insignificant 31 .…”

“…Previously it has been reported that PEG 4 kDa of small concentrations has little change without any shift in Soret absorption and oxy-deoxy band in cyt c 31 . The studies claim that PEG 4 kDa promotes auto-oxidation of cyt c , however changes are observed to be insignificant 31 . In addition, the study proposed a model showing that heme is dislocated due to dehydration of some surface exposed hydrophobic residues i.e., ILE81 and VAL83; however spectroscopic techniques showed small changes 31 and are comparable to our results at lower concentrations of the crowder.…”

“…Moreover, it is known that PEG’s are crystallizing agents 11 , precautions should be taken whilst using PEG’s as crystallizers because PEG is also a destabilizing agent at higher concentrations 4 , 5 , 16 , 27 , 28 . It should be noted that most of the PEGs and other crowders on interaction disrupts heme in the proteins (cytochrome c and myoglobin) 4 , 5 , 10 , 29 – 31 . Moreover, the effect of PEG 4 kDa at small concentrations on cyt c has been reported where it was observed that PEG 4 kDa increases the auto-oxidation of cyt c , however changes are insignificant 31 .…”

“…It should be noted that most of the PEGs and other crowders on interaction disrupts heme in the proteins (cytochrome c and myoglobin) 4 , 5 , 10 , 29 – 31 . Moreover, the effect of PEG 4 kDa at small concentrations on cyt c has been reported where it was observed that PEG 4 kDa increases the auto-oxidation of cyt c , however changes are insignificant 31 . In addition, the study proposed a model showing that heme is dislocated due to dehydration of some surface exposed hydrophobic residues i.e.…”