Local Conformational Transition of Hydrogenobacter thermophilus Cytochrome c552 Relevant to Its Redox Potential,

Takayama, Shinichi; Takahashi, Yurika; Mikami, Shuji; Irie, Kiyofumi; Kawano, Shin; Yamamoto, Yasuhiko; Hemmi, Hikaru; Kitahara, Ryo; Yokoyama, Shigeyuki; Akasaka, Kazuyuki

doi:10.1021/bi7000714

Cited by 10 publications

(28 citation statements)

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“…32 Assignments of ferrous Ht WT at 40 °C (pH 7.0) were made using TOCSY, NOESY, and 3-D HSQC-TOCSY spectra and by comparison to previous assignments both at 25 °C (pH 5) 32 and at 23 °C (pH 6). 54 Pseudocontact shifts were determined by taking the difference between the shift in the oxidized and reduced states, using nuclei for which contact shift is negligible (i.e., excluding the heme, Cys12, Cys15, His16, and Met61). A total of 51 pseudocontact shifts were used in the fitting along with the resonance assignments in both oxidation states (Table S4).…”

Section: Methodsmentioning

confidence: 99%

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

2013

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The heme in cytochromes c undergoes a conserved out-of-plane distortion known as ruffling. For cytochromes c from the bacteria Hydrogenobacter thermophilus and Pseudomonas aeruginosa, NMR and EPR spectra have been shown to be sensitive to the extent of heme ruffling and to provide insights into the effect of ruffling on electronic structure. Using mutants of each of these cytochromes that differ in the amount of heme ruffling, NMR characterization of the low-spin (S=1/2) ferric proteins has confirmed and refined the developing understanding of how ruffling influences spin distribution on heme. The chemical shifts of the core heme carbons were obtained through site-specific labeling of the heme via biosynthetic incorporation of 13C-labeled 5-aminolevulinic acid derivatives. Analysis of the contact shifts of these core heme carbons allowed Fermi contact spin densities to be estimated, and changes upon ruffling to be evaluated. The results allow a deconvolution of contributions to heme hyperfine shifts and a test of the influence of heme ruffling on electronic structure and hyperfine shifts. The data indicate that as heme ruffling increases, the spin densities on the β-pyrrole carbons decrease, while the spin densities on the α-pyrrole carbons and meso carbons increase. Furthermore, increased ruffling is associated with stronger bonding to the heme axial His ligand.

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Section: Methodsmentioning

confidence: 99%

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

2013

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“…WT Ht cyt c 552 has a fluxional axial Met, while the S and R configurations can be stabilized by the Q64N28 and Q64V29 mutations, respectively. An axial Met configuration change may also be correlated to other conformational changes of the axial Met-bearing loop 30. Based upon sequence and structural alignment of Ht cyt c 552 with Pa cyt c 551 , and by analogy to the enhanced ruffling in F7A Pa cyt c 551 , the A7F mutation is expected to reduce the magnitude of heme ruffling as compared to WT Ht cyt c 552 .…”

Section: Introductionmentioning

confidence: 99%

NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c

2010

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Out-of-plane (OOP) deformations of the heme cofactor are found in numerous heme-containing proteins and the type of deformation tends to be conserved within functionally-related classes of heme proteins. We demonstrate correlations between the heme ruffling OOP deformation and the 13 C and 1 H nuclear magnetic resonance (NMR) hyperfine shifts of heme aided by density functional theory (DFT) calculations. The degree of ruffling in the heme cofactor of Hydrogenobacter thermophilus cytochrome c 552 has been modified by a single amino acid mutation in the second coordination sphere of the cofactor. The 13 C and 1 H resonances of the cofactor have been assigned using one-and two-dimensional NMR spectroscopy aided by selective 13 C-enrichment of the heme. DFT has been used to predict the NMR hyperfine shifts and electron paramagnetic resonance (EPR) g-tensor at several points along the ruffling deformation coordinate. The DFT-predicted NMR and EPR parameters agree with the experimental observations, confirming that an accurate theoretical model of the electronic structure and its response to ruffling has been established. As the degree of ruffling increases, the heme methyl 1 H resonances move upfield while the heme methyl and meso 13 C resonances move downfield. These changes are a consequence of altered overlap of the Fe 3d and porphyrin π orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the β-pyrrole and meso carbons, respectively. Consequently, the heme ruffling deformation decreases the electronic coupling of the cofactor with external redox partners and lowers the reduction potential of heme.

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“…The backbone amide resonances of the wild-type protein were assigned using a 3D 15 N-edited NOESY-HSQC experiment with a mixing time of 75 ms (Marion et al 1989 ), with reference to previous assignments (Hasegawa et al 1998 ; Takayama et al 2007 ; Wain et al 2004 ). Five of the backbone resonance assignments of the b -type variant were revised following further analysis of the spectra (for Gln 3, Leu 4, Lys 36, Ser 51 and Met 59).…”

Section: Methodsmentioning

confidence: 99%

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

et al. 2015

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Cytochrome c552 from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, 15N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high 15N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

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Local Conformational Transition of Hydrogenobacter thermophilus Cytochrome c₅₅₂ Relevant to Its Redox Potential^,

Cited by 10 publications

References 50 publications

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

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Local Conformational Transition of Hydrogenobacter thermophilus Cytochrome c552 Relevant to Its Redox Potential,

Cited by 10 publications

References 50 publications

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core 13C NMR

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core 13C NMR

NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

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Local Conformational Transition of Hydrogenobacter thermophilus Cytochrome c₅₅₂ Relevant to Its Redox Potential^,

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR