Thermodynamics and Kinetics of Formation of the Alkaline State of a Lys 79→Ala/Lys 73→His Variant of Iso-1-cytochrome <i>c</i>

Baddam, Saritha; Bowler, Bruce E.

doi:10.1021/bi0515873

Cited by 34 publications

(150 citation statements)

References 51 publications

Supporting

Mentioning

136

Contrasting

Order By: Relevance

“…Based on previous work on K73H variants of iso-1-Cyt c , these phases likely correspond to formation of a Lys79-heme ligated conformer as well as isomerization of proline in the His73-heme alkaline conformer [29, 30, 42]. The observed rate constant for the first slow phase, k obs,3 (Fig.…”

Section: Resultsmentioning

confidence: 75%

See 1 more Smart Citation

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

et al. 2015

Self Cite

View full text Add to dashboard Cite

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70 to 85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer.

show abstract

Section: Resultsmentioning

confidence: 75%

“…Titrations were followed at 695 nm, as previously reported [42], in 100 mM NaCl using a Beckman Coulter DU 800 UV/Vis spectrophotometer. 600 μL of a 2x stock solution containing 400 μM oxidized protein in 200 mM NaCl was prepared.…”

Section: Methodsmentioning

confidence: 99%

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…25 However, the heme iron reduction potential remains largely unaffected in K79A 15 (Table 2) and the rate of the forward alkaline transition increases rather than decreases. 25, 99 Like K79G, K79A has an uncharged residue at position 79 with a relatively small side chain; however, alanine is less flexible than glycine, and its side chain is more hydrophobic. 100 Our modeling suggests distinct differences in the shape and solvation of the heme crevice between K79G and K79A.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

“…pH titrations in 0.5 M NaCl were also carried out. Titrations were carried out at 22 ± 1 °C using a Beckman DU 800 spectrophotometer, as previously described (24, 26, 28). Loss of heme-Met80 ligation was monitored at 695 nm (12).…”

Section: Methodsmentioning

confidence: 99%

“…Hexaammineruthenium(II) chloride (a 6 Ru 2+ ) was prepared by reducing commercially available [Ru(NH 3 ) 6 ]Cl 3 (Strem Chemicals) with zinc by the method of Fergusson and Love (52) and characterized by IR spectroscopy (53), as previously described (24, 26). …”

Section: Methodsmentioning

confidence: 99%

Effect of an Ala81His Mutation on the Met80 Loop Dynamics of Iso-1-cytochromec

Bandi

Bowler

2015

Biochemistry

Self Cite

View full text Add to dashboard Cite

An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric size and to provide a probe of the dynamics of the heme crevice through measurement of the thermodynamics and kinetics of the His81-mediated alkaline conformational transition of A81H iso-1-cytochrome c. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome c. ΔGuo(H2O) is about 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome c the native conformer is less stable than the His79-heme alkaline conformer. ΔGuo(H2O) is about −0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonstrate that this stabilization of the native conformer in A81H iso-1-cytochrome c arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, kf,His81, relative to kf,His79 for formation of the His79-heme alkaline conformer, which forms by a similar mechanism to that observed for the His81-heme alkaline conformer. The result is discussed in terms of the effect of global protein stability on protein dynamics and in terms of optimization of the sequence of cytochrome c for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes.

show abstract

Thermodynamics and Kinetics of Formation of the Alkaline State of a Lys 79→Ala/Lys 73→His Variant of Iso-1-cytochrome c

Cited by 34 publications

References 51 publications

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

The response of Ω-loop D dynamics to truncation of trimethyllysine 72 of yeast iso-1-cytochrome c depends on the nature of loop deformation

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

Effect of an Ala81His Mutation on the Met80 Loop Dynamics of Iso-1-cytochromec

Contact Info

Product

Resources

About