The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochromec

Abstract

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (pKa) of the protein. However, the reduction potential of the heme iron decreases; further, the pKH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively-charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T

“…This ligation switch is supported by RR data37 as well as NMR and EPR experiments 38. While the enhanced peroxidase activity of alkaline Lys–Fe conformers is well documented,38 the origin of this effect is somewhat unclear because transient opening of the Lys–Fe contact takes place more slowly than for Met–Fe 34,77. Likely, the enhanced activity of fraction I is not caused by hexacoordinated Lys–Fe species per se , but by pentacoordinated structures that are part of the fraction I ensemble 14,38.…”

“…37). Other factors may contribute to the peroxidase activity of Lys-ligated cyt c as well,41 one of these is the increased heme pocket volume which facilitates substrate access 19,77…”

Mechanistic Studies of Proton-Coupled Electron Transfer in a Calorimetry Cell

“…Our ITC-derived thermodynamic parameters for heme iron reduction are in accord with those from electrochemistry experiments for these and similarly ligated cyt c variants (Table S2). ,− …”

A Heme Propionate Staples the Structure of Cytochromecfor Methionine Ligation to the Heme Iron

“…Herein, we systematically compare effects of Thr to Val substitutions at residues 49 and 78 on the conformational and redox properties of cyt c . Our recently developed K79G variant of yeast iso -1 cyt c simplifies the study of the ligand-switch reactions, as only Lys73 can replace Met80 at the heme iron, and we incorporate this background substitution into two cyt c variants, T49V/K79G and T78V/K79G, to target the environment near HP6. Both Thr to Val substitutions modify reduction potentials and ligand-switch properties of cyt c , but their effects differ.…”