The hydrogen peroxide induced radical behaviour in human cytochrome c phospholipid complexes: Implications for the enhanced pro-apoptotic activity of the G41S mutant

DOI: 10.1042/BJ20130758
PMID: 24099549

Authors: Badri Rajagopal (University of Essex) , Ann Edzuma (University of Essex) , Mike Hough (University of Essex) , Katie Blundell (University of Essex) , Valerian Kagan (University of Pittsburgh) , Alexandr Kapralov (University of Pittsburgh) , Lewis Fraser (University of East Anglia) , Julea Butt (University of East Anglia) , Gary Silkstone (University of Essex) , Mike Wilson (University of Essex) , Dimitri Svistunenko (University of Essex) , Jonathan Worrall (University of Essex)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal

State: Published (Approved)
Published: October 2013

Abstract: We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met80 ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr46 and Tyr48, have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c–phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis

Journal Keywords: cardiolipin; cytochrome c; peroxidase; phospholipid; tyrosyl radical

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Added On: 12/11/2013 13:23

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)