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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.


(A) Superposition of 1H-15N HSQC spectra of ferrous cytochrome c3 at various pH values from pH 6.5 (yellow) through 7.0 (black) to 9.0 (purple). The direction of an arrow indicates the increase of pH value. (B) Average chemical shift differences (Δδave) between pH 7.0 and 8.5. Open circles indicate the residues whose NH signals were not detected at pH 8.5 although assigned at pH 7.0.
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f2-2_45: (A) Superposition of 1H-15N HSQC spectra of ferrous cytochrome c3 at various pH values from pH 6.5 (yellow) through 7.0 (black) to 9.0 (purple). The direction of an arrow indicates the increase of pH value. (B) Average chemical shift differences (Δδave) between pH 7.0 and 8.5. Open circles indicate the residues whose NH signals were not detected at pH 8.5 although assigned at pH 7.0.

Mentions: On the other hand, the spectra became more complicated in the reduced state compared to those of the oxidized one. In lower pH range than 6.5, many signals derived from the three-electron-reduced state (S3) and/or two-electron reduced state (S2) appeared in the spectra because of the shift of the redox-equilibrium between cyt c3 and hydrogenase. Therefore, the superposition of the HSQC spectra in the reduced state at the pH range from 6.5 to 9.0 is shown in Figure 2A. Furthermore, the average chemical shift differences (Δδave) of each backbone amide between pH 7.0 and 8.5 were shown in Figure 2B. The residues whose NH signals indicate larger differences (Δδave≥0.09) are Leu9, Lys10, Lys45, Cys46, Lys63, Tyr66, His67, Ala68, Asp71, and Phe76. These residues were mapped on the solution structure (Fig. 3B).


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
(A) Superposition of 1H-15N HSQC spectra of ferrous cytochrome c3 at various pH values from pH 6.5 (yellow) through 7.0 (black) to 9.0 (purple). The direction of an arrow indicates the increase of pH value. (B) Average chemical shift differences (Δδave) between pH 7.0 and 8.5. Open circles indicate the residues whose NH signals were not detected at pH 8.5 although assigned at pH 7.0.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036644&req=5

f2-2_45: (A) Superposition of 1H-15N HSQC spectra of ferrous cytochrome c3 at various pH values from pH 6.5 (yellow) through 7.0 (black) to 9.0 (purple). The direction of an arrow indicates the increase of pH value. (B) Average chemical shift differences (Δδave) between pH 7.0 and 8.5. Open circles indicate the residues whose NH signals were not detected at pH 8.5 although assigned at pH 7.0.
Mentions: On the other hand, the spectra became more complicated in the reduced state compared to those of the oxidized one. In lower pH range than 6.5, many signals derived from the three-electron-reduced state (S3) and/or two-electron reduced state (S2) appeared in the spectra because of the shift of the redox-equilibrium between cyt c3 and hydrogenase. Therefore, the superposition of the HSQC spectra in the reduced state at the pH range from 6.5 to 9.0 is shown in Figure 2A. Furthermore, the average chemical shift differences (Δδave) of each backbone amide between pH 7.0 and 8.5 were shown in Figure 2B. The residues whose NH signals indicate larger differences (Δδave≥0.09) are Leu9, Lys10, Lys45, Cys46, Lys63, Tyr66, His67, Ala68, Asp71, and Phe76. These residues were mapped on the solution structure (Fig. 3B).

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.