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Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F

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ABSTRACT

Complicated pH-properties of the tetraheme cytochrome c3 (cyt c3) from Desulfovibrio vulgaris Miyazaki F (DvMF) were examined by the pH titrations of 1H-15N HSQC spectra in the ferric and ferrous states. The redox-linked pKa shift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pKa shift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cyt c3 is similar to that of wild type, local change was observed in 1H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties of DvMF cyt c3. In contrast, the kinetic parameters for electron transfer from DvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.

No MeSH data available.


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Mappings of the residues showing large pH-dependent shifts on the cytochrome c3 structures. The regions around heme 1 (left) and heme 2 (right) in the ferric crystal structure (A; PDB code, 1J0O) and the ferrous solution structure (model 1) (B; PDB code, 1IT1). Residues are colored according to the following classification: (A) ΔΔave (pH 5.2–7.0)>0.15 ppm (magenta), Δδave (pH 7.0–8.5)>0.15 ppm (cyan); (B) Δδave (pH 7.0–8.5)>0.09 ppm (cyan). Focused heme is colored red. These figures were produced with the Chimera program37.
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f3-2_45: Mappings of the residues showing large pH-dependent shifts on the cytochrome c3 structures. The regions around heme 1 (left) and heme 2 (right) in the ferric crystal structure (A; PDB code, 1J0O) and the ferrous solution structure (model 1) (B; PDB code, 1IT1). Residues are colored according to the following classification: (A) ΔΔave (pH 5.2–7.0)>0.15 ppm (magenta), Δδave (pH 7.0–8.5)>0.15 ppm (cyan); (B) Δδave (pH 7.0–8.5)>0.09 ppm (cyan). Focused heme is colored red. These figures were produced with the Chimera program37.

Mentions: The pH titrations of 1H-15N HSQC spectra for the ferric and ferrous cyt c3 were performed in the pH range of 5.2–9.0. The superposition of the HSQC spectra in the oxidized state acquired at various pH values are shown in Figure 1A. Several signals indicated distinct transitions either in the acidic or basic pH region. In a pH range lower than 6, two non-assigned signals, named “a” (1H, 8.72 ppm; 15N, 109.1 ppm) and “b” (8.15; 124.5) are observed (Fig. 1A). These signals could be ascribed to non-assigned backbone amides, Ala1 and Gly73. The NH signal of Gly73 in the reduced cyt c3 was assigned (1H, 8.92 ppm; 15N, 109.6 ppm)17. Signal “a” can be ascribed to Gly73 based on the assignment in the reduced state and the averaged 15N amide chemical shift data of each amino acid residue32. The appearance of Ala1 amide signal clearly indicates the presence of another alanine residue at the N-terminus as previously reported33. To estimate the effects of the pH changes on the properties of cyt c3, the average chemical shift differences (Δδave) of each backbone amide between pH 5.2 and 7.0, and pH 7.0 and 8.5, were calculated using the equation, Δδave=[ΔδH2+(ΔδN/5)2]1/2, where ΔδH and ΔδN stand for the differences in the 1H and 15N chemical shifts, respectively (Figs. 1B and 1C). The residues whose NH signals indicated larger differences (Δδave≥0.15) in the acidic pH region were Glu41, Asp42, Gln44 (side chain NH), and Cys46. In the basic pH region, on the other hand, Lys63, His67, Ala68, Asp71, Thr74, and Phe76 indicated larger differences (Δδave≥0.15). These residues were mapped on the crystal structure (Fig. 3A). Additionally, the NH signal of His83 indicated strange transition (Fig. 1A).


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
Mappings of the residues showing large pH-dependent shifts on the cytochrome c3 structures. The regions around heme 1 (left) and heme 2 (right) in the ferric crystal structure (A; PDB code, 1J0O) and the ferrous solution structure (model 1) (B; PDB code, 1IT1). Residues are colored according to the following classification: (A) ΔΔave (pH 5.2–7.0)>0.15 ppm (magenta), Δδave (pH 7.0–8.5)>0.15 ppm (cyan); (B) Δδave (pH 7.0–8.5)>0.09 ppm (cyan). Focused heme is colored red. These figures were produced with the Chimera program37.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036644&req=5

f3-2_45: Mappings of the residues showing large pH-dependent shifts on the cytochrome c3 structures. The regions around heme 1 (left) and heme 2 (right) in the ferric crystal structure (A; PDB code, 1J0O) and the ferrous solution structure (model 1) (B; PDB code, 1IT1). Residues are colored according to the following classification: (A) ΔΔave (pH 5.2–7.0)>0.15 ppm (magenta), Δδave (pH 7.0–8.5)>0.15 ppm (cyan); (B) Δδave (pH 7.0–8.5)>0.09 ppm (cyan). Focused heme is colored red. These figures were produced with the Chimera program37.
Mentions: The pH titrations of 1H-15N HSQC spectra for the ferric and ferrous cyt c3 were performed in the pH range of 5.2–9.0. The superposition of the HSQC spectra in the oxidized state acquired at various pH values are shown in Figure 1A. Several signals indicated distinct transitions either in the acidic or basic pH region. In a pH range lower than 6, two non-assigned signals, named “a” (1H, 8.72 ppm; 15N, 109.1 ppm) and “b” (8.15; 124.5) are observed (Fig. 1A). These signals could be ascribed to non-assigned backbone amides, Ala1 and Gly73. The NH signal of Gly73 in the reduced cyt c3 was assigned (1H, 8.92 ppm; 15N, 109.6 ppm)17. Signal “a” can be ascribed to Gly73 based on the assignment in the reduced state and the averaged 15N amide chemical shift data of each amino acid residue32. The appearance of Ala1 amide signal clearly indicates the presence of another alanine residue at the N-terminus as previously reported33. To estimate the effects of the pH changes on the properties of cyt c3, the average chemical shift differences (Δδave) of each backbone amide between pH 5.2 and 7.0, and pH 7.0 and 8.5, were calculated using the equation, Δδave=[ΔδH2+(ΔδN/5)2]1/2, where ΔδH and ΔδN stand for the differences in the 1H and 15N chemical shifts, respectively (Figs. 1B and 1C). The residues whose NH signals indicated larger differences (Δδave≥0.15) in the acidic pH region were Glu41, Asp42, Gln44 (side chain NH), and Cys46. In the basic pH region, on the other hand, Lys63, His67, Ala68, Asp71, Thr74, and Phe76 indicated larger differences (Δδave≥0.15). These residues were mapped on the crystal structure (Fig. 3A). Additionally, the NH signal of His83 indicated strange transition (Fig. 1A).

View Article: PubMed Central - PubMed

ABSTRACT

Complicated pH-properties of the tetraheme cytochrome c3 (cyt c3) from Desulfovibrio vulgaris Miyazaki F (DvMF) were examined by the pH titrations of 1H-15N HSQC spectra in the ferric and ferrous states. The redox-linked pKa shift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pKa shift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cyt c3 is similar to that of wild type, local change was observed in 1H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties of DvMF cyt c3. In contrast, the kinetic parameters for electron transfer from DvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.

No MeSH data available.


Related in: MedlinePlus