Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions

Lett, C. Marc; Guillemette, J. Guy

doi:10.1042/bj3620281

Cited by 22 publications

(19 citation statements)

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“…14,24,85 By focusing on alterations of these properties in cyt c variants that carry replacements in the crevice loop sequence, one can rationalize the role of this loop and cyt c structure in governing the redox functions of cyt c. The decrease in reduction potential in our K79G mutant compared to the parent WT* is relatively small and falls within the range of changes reported for other mutations of Lys79 to a small neutral residue (Table 2). 15,70,86 However, we do observe notable changes in several kinetic assays with K79G suggesting that alterations in the structure and dynamics of the heme crevice occur upon replacing Lys79. Structural modeling as well as analyses of global stability and local dynamics of the heme crevice loop upon this mutation provide clues regarding the origin of these effects and shed light on the role of Lys79.…”

Section: ■ Discussionmentioning

confidence: 68%

“…15,58,76 Considering the experimental uncertainty of the cyclic voltammetry measurements (Table 2), the reduction potential of WT* does not appear to change significantly from the value of WT measured under the same conditions. 15,70,86 These findings suggest that structural and dynamic alterations near the heme upon the tmK72A mutation are likely minor, but the details of these perturbations have remained unknown. A crystal structure of the hydroxide-ligated WT* has revealed an extensive buried water channel.…”

Section: ■ Discussionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: ■ Discussionmentioning

confidence: 68%

Section: ■ Discussionmentioning

confidence: 99%

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

et al. 2018

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“…32 The yeast iso-cyt c variant Y48H was reported to exhibit a decreased midpoint potential (approximately −80 mV compared to wild-type iso-cyt c). 33 A complete biophysical characterization of human and yeast counterparts of variant Y48H is required to fully comprehend their behavior toward CL binding, electron transfer, and other redox processes.…”

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confidence: 98%

Alternative Conformations of Cytochrome c: Structure, Function, and Detection

et al. 2016

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Cytochrome c (cyt c) is a cationic hemoprotein of ∼100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery, and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations, and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their functions in vivo continue to be a major challenge. Here, we unify the knowledge of the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.

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“…While structural data exist only for Thr49 proteins, presumably both amino acids could engage in hydrogen bonding with HP6. Before our work, there has been only one report of a Thr49 mutant . The yeast iso-1 cyt c T49K mutant has been shown to support oxidative phosphorylation in yeast cells deficient in their ability to produce other isoforms of cyt c , but no biophysical characterization of this variant has been performed.…”

Section: Discussionmentioning

confidence: 99%

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochromec

Shin

Pletneva

2017

Biochemistry

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Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), Met80 ligation to the heme iron is critical for the protein’s electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, NMR, and EPR spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation and the pH-dependent changes in ligation provide detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling the native Met80 ligation to the heme iron.

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Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions

Cited by 22 publications

References 0 publications

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

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

Alternative Conformations of Cytochrome c: Structure, Function, and Detection

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochromec

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