Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity

Haotian Lei; Bruce E. Bowler

doi:10.1021/acs.jpcb.9b05869

Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity

Haotian Lei
, Bruce E. Bowler

Chemistry and Biochemistry

University of Montana

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH_1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH_1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the k_cat values for its peroxidase activity increase by 6-to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.

Original language	English
Pages (from-to)	8939-8953
Number of pages	15
Journal	Journal of Physical Chemistry B
Volume	123
Issue number	42
DOIs	https://doi.org/10.1021/acs.jpcb.9b05869
State	Published - Oct 24 2019

Funding

This work was supported by NSF Grant CHE-1609720 (B.E.B). The Macromolecular X-ray Diffraction Core Facility at the University of Montana was supported by a CoBRE grant from the National Institute of General Medical Sciences (P20GM103546). We thank the staff at the Stanford Synchrotron Radiation Lightsouce (SSRL) SMB for assistance with data collection. Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under Contract DEAC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including Grant P41GM103393).

Funders	Funder number
DEAC02-76SF00515
CHE-1609720
P41GM103393
P20GM103546
SLAC National Accelerator Laboratory

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title = "Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity",

abstract = "The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 {\AA} structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the kcat values for its peroxidase activity increase by 6-to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.",

author = "Haotian Lei and Bowler, \{Bruce E.\}",

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N2 - The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the kcat values for its peroxidase activity increase by 6-to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.

AB - The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the kcat values for its peroxidase activity increase by 6-to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis.

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Naturally Occurring A51V Variant of Human Cytochrome c Destabilizes the Native State and Enhances Peroxidase Activity

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