Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity

Biochemistry. 2017 Nov 21;56(46):6111-6124. doi: 10.1021/acs.biochem.7b00890. Epub 2017 Nov 8.


Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40-57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome c fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 "off rate" of >125 s-1 compared to a rate of ∼6 s-1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40-57 Ω-loop and the Met80-containing 71-85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome c variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome c is emerging whereby increasing dynamics in substructures of the cytochrome c fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Cytochromes c / chemistry
  • Cytochromes c / genetics*
  • Cytochromes c / metabolism*
  • Enzyme Stability
  • Heme / chemistry
  • Heme / genetics
  • Heme / metabolism
  • Humans
  • Molecular Dynamics Simulation
  • Oxidation-Reduction
  • Peroxidase / chemistry
  • Peroxidase / genetics
  • Peroxidase / metabolism
  • Point Mutation*
  • Protein Conformation
  • Protein Folding
  • Thermodynamics


  • Heme
  • Cytochromes c
  • Peroxidase