Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome <i>c</i>

Nold, Shiloh M.; Lei, Haotian; Mou, Tung-Chung; Bowler, Bruce E.

doi:10.1021/acs.biochem.7b00342

Cited by 31 publications

(76 citation statements)

References 73 publications

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“…87 Similar global stabilities, apparent p K a and kinetic parameters of alkaline transition for yeast WT and WT* reiterate previous conclusions that steric hindrance within the heme crevice loop is minimal in the yeast protein. 87 Together these results imply that Lys72 plays a less important role in determining functional properties of yeast cyt c compared to its mammalian counterparts.…”

Section: Discussionsupporting

confidence: 77%

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: Discussionsupporting

confidence: 77%

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

et al. 2018

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“…This triggers conversion of the Fe to the active pentacoordinate state, and once in this state the cytochrome c variants have the same activity. A possible explanation for the increased activity of the mutations is that they enhance accessibility of the heme to peroxide, and previous studies have suggested that rigidity of the 71-85 Ω loop acts to inhibit peroxidase activity (43). Our extensive MD data with additional variants does not support a hypothesis that increased movement of the cytochrome c main chain, particularly in the 71-85 Ω loop, enhances conversion to the active state.…”

Section: Discussioncontrasting

confidence: 78%

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

Fellner

Parakra

McDonald

et al. 2020

Preprint

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AbstractMutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with exchange between conformational states proposed to regulate changes in function. Here we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. We find that three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with increased overall flexibility of cytochrome c, particularly movement of the Ω loops. This suggests that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an “induced fit” mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics suggesting that the mechanism by which the variants alter peroxidase activity is not related to the conformation dynamics of the hexacoordinate heme Fe state of cytochrome c analyzed in the simulations. Recent suggestions that conformational mobility of specific regions of cytochrome c underpins changes in reduction potential and the alkaline transition pK were not supported. These data highlight that conformational dynamics of cytochrome c drives some but not all of its properties and activities.

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“…And the involvement of Pro71 (CH-p interaction with a phenol ring) is also reminiscent of the PAF-sclx 6 complexation (compare Figs 1 and 2C). Calix [6]arene complexation and cytc restructuring is interesting in the context of the alkaline conformational transition, which can involve Lys72, Lys73, or Lys79 on Ω-loop D [8,[10][11][12][13][14]. Apparently, the mobility of this loop facilitates the two different processes.…”

Section: Resultsmentioning

confidence: 99%

Calixarene capture of partially unfolded cytochrome c

2019

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Supramolecular receptors such as water‐soluble calixarenes are in development as ‘molecular glues’ for protein assembly. Here, we obtained cocrystals of sulfonato‐calix[6]arene (sclx6) and yeast cytochrome c (cytc) in the presence of imidazole. A crystal structure at 2.65 Å resolution reveals major structural rearrangement and disorder in imidazole‐bound cytc. The largest protein‐calixarene interface involves 440 Å2 of the protein surface with key contacts at Arg13, Lys73, and Lys79. These lysines participate in alkaline transitions of cytc and are part of Ω‐loop D, which is substantially restructured in the complex with sclx6. The structural modification also includes Ω‐loop C, which is disordered (residues 41–55 inclusive). These results suggest the possibility of using supramolecular scaffolds to trap partially disordered proteins.

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Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c

Cited by 31 publications

References 73 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

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

Calixarene capture of partially unfolded cytochrome c

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