Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity

Lei, Haotian; Bowler, Bruce E.

doi:10.1016/j.jinorgbio.2018.02.022

Cited by 9 publications

(38 citation statements)

References 63 publications

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“…We initially tested this hypothesis with A81I and G83V substitutions to yeast iso-1-Cytc. 23 We observed small decreases in the peroxidase activity of iso-1-Cytc, consistent with our hypothesis. However, these decreases were small relative to the large decrease in the peroxidase activity of Hu Cytc relative to yeast iso-1-Cytc.…”

supporting

confidence: 91%

“…The G83V variant of iso-1-Cytc also shows a loss of cooperativity for acid unfolding (acid unfolding monitored at 695 nm is biphasic). 23 Neither variant of Hu Cytc shows biphasic acid unfolding when monitored at 695 nm (Figure 4). Studies on disease-causing variants on Hu Cytc suggest that these context-dependent effects may involve communication between Ω-loops C and D, 32 mediated by the more substantial sequence divergence of Ω-loop C for the yeast versus the human protein.…”

Section: ■ Discussionmentioning

confidence: 99%

“…49 pH Jump Stopped-Flow Kinetics of the Alkaline Transition. As previously reported, 23,24 pH jump stoppedflow experiments were executed at 25 °C using an Applied Photophysics SX20 stopped-flow spectrometer. A total of 5000 data points were collected on a logarithmic time scale monitoring at 398 nm (A 398 ).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…59 The alkaline conformer of Hu Cytc is much more accessible (lower pK app ) for both the G41S and Y48H disease-causing variants. 5,6,29,30,33 Often a good correlation between peroxidase activity and the pK app for the alkaline transition is observed, 23 but the correlation is not perfect. 32 Thus, we measured the local unfolding of Hu Cytc variants at alkaline pH at 695 nm to observe the loss of Met80heme ligation.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…69 The cooperativity of acid unfolding can readily be disrupted by amino acid substitutions. 23,46,48,70 In some cases, a loss of cooperativity may reflect an increase in the favorability of a low spin His-heme state II conformer over the high spin state II conformer. 68 Acid unfolding data presented here are carried out in 0.1 M NaCl.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Effect of V83G and I81A Substitutions to Human Cytochrome c on Acid Unfolding and Peroxidase Activity below a Neutral pH

et al. 2019

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Mitochondrial cytochrome c is a highly conserved protein in eukaryotes. Certain functions of cytochrome c have been tuned during evolution. For instance, the intrinsic peroxidase activity of human cytochrome c is much lower than that of the yeast counterpart. Structural studies on K72A yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. USA, 111, 6648–6653] revealed that residues 81 and 83 in Ω-loop D (residues 70–85) may be gatekeeper residues for the peroxidase activity linked to intrinsic apoptosis. Amino acids at both positions evolve to more sterically demanding amino acids. We hypothesized that residues 81 and 83 evolved such that steric constraints at these positions tune down the peroxidase activity of human cytochrome c. To test this hypothesis, I81A and V83G variants of human cytochrome c were prepared. Our results show that the I81A substitution significantly influences the thermodynamics and kinetics of access to alternate conformers of human cytochrome c, while the V83G substitution has a more modest effect on these properties. The I81A variant also shows a significant enhancement in peroxidase activity, particularly below pH 7, whereas the V83G variant has a similar peroxidase activity to wild-type human cytochrome c. However, neither variant increases the peroxidase activity of human cytochrome c to the level of yeast iso-1-cytochrome c, indicating that other substructures of cytochrome c are also involved in tuning the intrinsic peroxidase activity of mitochondrial cytochrome c.

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supporting

confidence: 91%

Section: ■ Discussionmentioning

confidence: 99%