Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

Sakamoto, Koichi; Kamiya, Masakatsu; Uchida, Takeshi; Kawano, Keiichi; Ishimori, Koichiro

doi:10.1016/j.bbrc.2010.06.065

Cited by 16 publications

(19 citation statements)

References 26 publications

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“…The reduced effect of the fast relaxation indicates a weaker association of oxidized Cyt c with CcO after electron transfer to CcO has occurred. Redox-dependent changes in the dynamic behavior of the N-terminal helix significantly more intense than that of the C-terminals are unlikely to be induced by the intrinsic properties of the Cyt c protein, because the redoxdependence of order parameter (S 2 ) of the N-terminal helix of free Cyt c in solution is not prominent, compared with other regions (32) (Fig. S7).…”

Section: Comparisons Of the Present Results With Other Electron Transmentioning

confidence: 99%

“…A gene encoding human Cyt c was synthesized using a modified recursive PCR strategy. Both genes of human Cyt c and yeast Cyt c heme lyase, which form the thioether bonds necessary for the maturation of Cyt c in the host cell (32), were cloned into the pET-21 (þ) vector using SacI and HindIII restriction sites. The protein expression and purification were performed according to previously published procedures (32).…”

Section: Methodsmentioning

confidence: 99%

“…Both genes of human Cyt c and yeast Cyt c heme lyase, which form the thioether bonds necessary for the maturation of Cyt c in the host cell (32), were cloned into the pET-21 (þ) vector using SacI and HindIII restriction sites. The protein expression and purification were performed according to previously published procedures (32). Purified protein with an R-value (¼A 409 ∕A 280 ) over 4.5 was collected to dissolve into 50 mM sodium phosphate buffer at pH 7.0 by centrifugal ultrafiltration.…”

Section: Methodsmentioning

confidence: 99%

“…The reduced form of Cyt c was obtained by anaerobically adding the sodium dithionite solution to the oxidized Cyt c solution. All NMR experiments were performed at 298 K on a Bruker DRX600 spectrometer equipped with a cryoprobe (32). All spectra were processed by NMR pipe (34) and the NMR data were analyzed with SPARKY (Goddard TD and Kneller DG, SPARKY 3, University of California, San Francisco http://www.cgl.ucsf.edu/home/sparky/).…”

Section: Methodsmentioning

confidence: 99%

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NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

Sakamoto

Kamiya²,

Imai³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c ) to cytochrome c oxidase (C c O) is investigated by 1 H- 15 N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled C c O indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c , the specific line broadening of N-H signals disappeared and high field 1 H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with C c O are reduced by steric constraint in oxidized Cyt c , while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with C c O. These results suggest that the overall affinity of oxidized Cyt c for C c O is significantly, but not very much weaker than that of reduced Cyt c . Thus, electron transfer is gated by dissociation of oxidized Cyt c from C c O, the rate of which is controlled by the affinity of oxidized Cyt c to C c O for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon C c O binding to oxidized Cyt c , shown by 1 H- and 1 H, 15 N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.

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Section: Comparisons Of the Present Results With Other Electron Transmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

Sakamoto

Kamiya²,

Imai³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…This agreement between the free energy for the Cyt c-CcO complex formation in the equilibrium state and that for the formation of the kinetic intermediate indicates that the complex structure under turnover conditions is quite similar to that in the equilibrium state. Such structural similarity between the two states is supported by the rigid dynamic structure of Cyt c (46,52). The generalized order parameters (s 2 ), the effective correlation time for internal motion ( e ), the 15 N exchange broadening contributions (R ex ) for each residue, and the overall correlation time ( m ) clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core (46).…”

Section: Energetic Analysis Of Interactions Between Cyt C and Ccomentioning

confidence: 91%

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

Sato

Hitaoka

Inoue

et al. 2016

Journal of Biological Chemistry

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Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. The electron transfer (ET)3 reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. A series of ET reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water. Associated with the reduction of molecular oxygen, CcO functions as a proton pump across the membrane, and the proton gradient is the primary driving force for the generation of ATP (1-5).In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at CcO are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c is thought to form an ET complex with CcO to promote the ET reaction from the heme iron in Cyt c to the Cu A site in CcO (6, 7). Although the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO regulate the binding affinity and the ET rate from Cyt c to CcO.The amino acid sequence and isoelectric point of Cyt c suggest that many positively charged residues are located on the protein surface (6, 7), as confirmed by solving the three-dimensional structures of Cyt c (8), allowing...

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Going with the Electron Flow: Heme Electronic Structure and Electron Transfer in Cytochrome c

Bren

2016

Israel Journal of Chemistry

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Heme is an essential and functionally versatile cofactor. Our understanding of how the environment of a heme in a protein tunes its function has benefited from spectroscopic and functional investigations of heme proteins and their variants with altered heme environments. Two properties of current interest are the conformation of the heme and hydrogen bonding to heme propionates. By combining nuclear magnetic resonance experiments and density functional theory calculations, both of these characteristics have been shown to influence the distribution of the singly occupied molecular orbital on the heme of ferricytochrome c, which affects coupling to redox partners and electron‐transfer rates. In addition, heme conformation has been shown to tune reduction potential. These results reveal that subtle variations in heme conformation and in interactions with its propionates can have significant impacts on electron‐transfer activity.

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Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

Cited by 16 publications

References 26 publications

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation

Going with the Electron Flow: Heme Electronic Structure and Electron Transfer in Cytochrome c

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