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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.

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p2H dependences of the heme methyl signals for wildtype and E41K cytochromes c3. (A) signal B for heme 1, (B) signal C for heme 2, (C) signal E for heme 3, and (D) signal A for heme 4. Diamonds, squares, triangles, and circles represent the fully oxidized (S0), one-electron-reduced (S1), two-electron-reduced (S2), and three-electron-reduced state (S3), respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
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f7-2_45: p2H dependences of the heme methyl signals for wildtype and E41K cytochromes c3. (A) signal B for heme 1, (B) signal C for heme 2, (C) signal E for heme 3, and (D) signal A for heme 4. Diamonds, squares, triangles, and circles represent the fully oxidized (S0), one-electron-reduced (S1), two-electron-reduced (S2), and three-electron-reduced state (S3), respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.

Mentions: The p2H titrations of heme methyl signals in each macroscopic oxidation state were performed for wild type, E41K, and K101M in p2H ranges of 4.9–9.7, 5.1–8.7, and 5.0–9.5, respectively. The chemical exchange spectra of wild type, E41K, and K101M were recorded at 600 MHz, 600 MHz, and 800 MHz, respectively. The signals in each p2H were assigned based on the chemical shifts at p2H 7.0 and previously reported data for wild type3. The obtained chemical shifts of the heme methyl signals B (heme 1), C (heme 2), E (heme 3), and A (heme 4) are plotted as a function of p2H. The titration curves for the wild type and E41K were presented in Figure 7. The titration curves for K101M were similar to those for the wild type. As reported previously, the methyl signals of heme 1 exhibited significant p2H dependence in the acidic pH region3. The midpoints of signal B in the one-electron reduced state determined by the nonlinear least-squares method were 5.6, 6.1, and 5.6 for wild type, E41K, and K101M, respectively. These results indicated that the E41K mutation induced a small shift in the titration curve toward high p2H. Furthermore, the complicated p2H dependences were observed in the titration curves for heme 3, especially in the two-electron reduced state, which might have something to do with the non-linear change in the chemical shift of the fifth axial ligand of heme 3, His83 (Fig. 1A).


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
p2H dependences of the heme methyl signals for wildtype and E41K cytochromes c3. (A) signal B for heme 1, (B) signal C for heme 2, (C) signal E for heme 3, and (D) signal A for heme 4. Diamonds, squares, triangles, and circles represent the fully oxidized (S0), one-electron-reduced (S1), two-electron-reduced (S2), and three-electron-reduced state (S3), respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
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f7-2_45: p2H dependences of the heme methyl signals for wildtype and E41K cytochromes c3. (A) signal B for heme 1, (B) signal C for heme 2, (C) signal E for heme 3, and (D) signal A for heme 4. Diamonds, squares, triangles, and circles represent the fully oxidized (S0), one-electron-reduced (S1), two-electron-reduced (S2), and three-electron-reduced state (S3), respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
Mentions: The p2H titrations of heme methyl signals in each macroscopic oxidation state were performed for wild type, E41K, and K101M in p2H ranges of 4.9–9.7, 5.1–8.7, and 5.0–9.5, respectively. The chemical exchange spectra of wild type, E41K, and K101M were recorded at 600 MHz, 600 MHz, and 800 MHz, respectively. The signals in each p2H were assigned based on the chemical shifts at p2H 7.0 and previously reported data for wild type3. The obtained chemical shifts of the heme methyl signals B (heme 1), C (heme 2), E (heme 3), and A (heme 4) are plotted as a function of p2H. The titration curves for the wild type and E41K were presented in Figure 7. The titration curves for K101M were similar to those for the wild type. As reported previously, the methyl signals of heme 1 exhibited significant p2H dependence in the acidic pH region3. The midpoints of signal B in the one-electron reduced state determined by the nonlinear least-squares method were 5.6, 6.1, and 5.6 for wild type, E41K, and K101M, respectively. These results indicated that the E41K mutation induced a small shift in the titration curve toward high p2H. Furthermore, the complicated p2H dependences were observed in the titration curves for heme 3, especially in the two-electron reduced state, which might have something to do with the non-linear change in the chemical shift of the fifth axial ligand of heme 3, His83 (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