Probing the Dynamics of a His73–Heme Alkaline Transition in a Destabilized Variant of Yeast Iso-1-cytochrome <i>c</i> with Conformationally Gated Electron Transfer Methods

Bandi, Swati; Bowler, Bruce E.

doi:10.1021/bi201082h

Cited by 18 publications

(95 citation statements)

References 79 publications

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“…The kinetic square-scheme used to evaluate gated ET involving reduction of A81H iso-1-Cyt c by hexaammineruthenium(II), a 6 Ru 2+ , is shown in Figure 8. In our experience, the more favorable heme reduction potential of the native conformer compared to alkaline conformers of iso-1-Cyt c (18, 77) makes path B the dominant path for reduction of the alkaline conformer by a 6 Ru 2+ (25–28, 30, 69). For reduction of the His81-heme alkaline conformer by a 6 Ru 2+ via path B, the rate constant for gated ET, k gET,2 , is given by eq 4 (78–80).…”

Section: Resultsmentioning

confidence: 87%

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

Bandi

Bowler

2015

Biochemistry

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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.

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Section: Resultsmentioning

confidence: 87%

“…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%

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

Bandi

Bowler

2015

Biochemistry

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“…Conformationally-gated electron transfer (gated ET) experiments were performed by following reduction of oxidized WT*/K73H Cyt c at 550 nm, a wavelength that is very sensitive to the redox state of Cyt c , with an Applied Photophysics SX20 stopped-flow spectrometer using previously established methods [9, 23, 29, 44]. The reducing reagent, hexaammineruthenium(II) chloride (a 6 Ru 2+ ), was prepared by reduction of hexaammineruthenium(III) chloride (Strem Chemicals) with zinc [45].…”

Section: Methodsmentioning

confidence: 99%

“…The alkaline conformational transition of wild-type iso-1-Cyt c expressed from yeast (yWT) is a monophasic conformational transition with either Lys73 or Lys79 replacing Met80 with similar, but not identical, kinetics and thermodynamics [18, 19]. Variants with a Lys73 to histidine mutation exhibit a biphasic alkaline transition where His73-heme ligation occurs near neutral pH, followed by Lys79-heme ligation above pH 8 [9, 20-22]. Not only does incorporating His into Ω-loop D enable the distinction between residues replacing Met80, but additionally the shift in the apparent p K a , p K app , of the alkaline transition allows the conformational change to be monitored more effectively because it can be done near neutral pH [20, 23].…”

Section: Introductionmentioning

confidence: 99%

“…In the case of Cyt c , the effect of the tmK72A mutation on Ω-loop D mediated from position 73 versus 79 could provide greater insight into the nature of the dynamics required for peroxidase function at the onset of apoptosis. Previous work has shown that the mechanisms of the His79-mediated alkaline transition and the His73-mediated alkaline transition differ [9, 21-23, 26, 27, 29, 30]. Additionally, denaturant m -values, which correlate to the surface area exposed by partial or full unfolding of a protein [15, 31, 32], are smaller for formation of the His79-heme alkaline conformer than for the His73-heme alkaline conformer indicating less structural rearrangement during formation of the His79-heme conformer than for the His73-heme conformer [20, 22, 23, 33].…”

Section: Introductionmentioning

confidence: 99%

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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

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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.

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A cytochrome C electron transfer switch modulated by heme ligation and isomerization of a peptidyl‐prolyl bond

Bandi

Bowler

2013

Biopolymers

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Intermolecular electron transfer (ET) between hexaamineruthenium(II), a(6) Ru(2+) , and a K73H/K79A variant of iso-1-cytochrome c, iso-1-Cytc, is used to study conformational ET switches mediated by His73-heme ligation and cis to trans isomerization of the Ile75-Pro76 peptidyl-prolyl bond of iso-1-Cytc. The biomolecular rate constant for ET to the native state of K73H/K79A iso-1-Cytc is ∼270 mM(-1) s(-1) near neutral pH. The unimolecular conformational ET switches due to His73-heme ligation and the Ile75-Pro76 peptidyl-prolyl bond gate ET at rate constants of 5 to 10 s(-1) and 0.05 to 0.06 s(-1) . Thus, at 1 mM a(6) Ru(2+) , these conformational ET switches slow electron transfer by about 50- and 5000-fold, respectively. The conformational ET switches are populated between pH 5 and 7, providing a means of modulating ET in this redox protein over several orders of magnitude by simply changing pH. The conformationally-gated ET measurements are analyzed in the context of previous pH jump measurements on the His73-heme alkaline transition of K73H/K79A iso-1-Cytc. The ability to obtain microscopic rate constants with conformationally-gated ET measurements has allowed more precise determination of the pK(a) s of the three ionizable groups that mediate population of the His73-heme ET switch. We have also been able to show that the ionizable group with a pK(a) near 9 stabilizes the His73-heme conformer relative to the native state of iso-1-Cytc and that contrary to the conclusions from our pH jump studies, this ionization does not strongly affect the rate of the Ile75-Pro76 peptidyl-prolyl isomerization.

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Probing the Dynamics of a His73–Heme Alkaline Transition in a Destabilized Variant of Yeast Iso-1-cytochrome c with Conformationally Gated Electron Transfer Methods

Cited by 18 publications

References 79 publications

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

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

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

A cytochrome C electron transfer switch modulated by heme ligation and isomerization of a peptidyl‐prolyl bond

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