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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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pH dependences of 15N chemical shifts of the NH signals for the ferric and ferrous cytochromes c3. Solid lines (closed signals) and broken ones (open ones) stand for the ferric and ferrous cytochromes c3, respectively. (A) NH signals for Glu41 and Asp42 stand for triangles and squares, respectively. (B) NH signals for His67, Ala68, Asp71, and Phe76 stand for squares, triangles, circles, and diamonds, respectively. (C) NH signals for Leu9, Lys10, Lys45, and Cys46 stand for triangles, squares, diamonds, and circles, respectively.
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f4-2_45: pH dependences of 15N chemical shifts of the NH signals for the ferric and ferrous cytochromes c3. Solid lines (closed signals) and broken ones (open ones) stand for the ferric and ferrous cytochromes c3, respectively. (A) NH signals for Glu41 and Asp42 stand for triangles and squares, respectively. (B) NH signals for His67, Ala68, Asp71, and Phe76 stand for squares, triangles, circles, and diamonds, respectively. (C) NH signals for Leu9, Lys10, Lys45, and Cys46 stand for triangles, squares, diamonds, and circles, respectively.

Mentions: The pH titration curves of the amide protons of Glu41 and Asp42 in the oxidized and reduced states are plotted in Figure 4A. For the ferrous cyt c3, it is assumed that NH signal shifts did not influenced by the redox-equilibrium in the acidic pH region. These titration curves in each redox state are similar, that is, pKas of these titration curves are not dependent on the redox states. For the residues indicated large NH signal changes in both states, His67, Ala68, Asp71, and Phe76, pH titration curves are also plotted in Figure 4B. These residues also show the similar pH dependences. On the other hand, the titration curves for Leu9, Lys10, Lys45, and Cys46 amide proton signals plotted in Figure 4C revealed different pH dependences between the oxidized and reduced states.


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
pH dependences of 15N chemical shifts of the NH signals for the ferric and ferrous cytochromes c3. Solid lines (closed signals) and broken ones (open ones) stand for the ferric and ferrous cytochromes c3, respectively. (A) NH signals for Glu41 and Asp42 stand for triangles and squares, respectively. (B) NH signals for His67, Ala68, Asp71, and Phe76 stand for squares, triangles, circles, and diamonds, respectively. (C) NH signals for Leu9, Lys10, Lys45, and Cys46 stand for triangles, squares, diamonds, and circles, respectively.
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Related In: Results  -  Collection

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

f4-2_45: pH dependences of 15N chemical shifts of the NH signals for the ferric and ferrous cytochromes c3. Solid lines (closed signals) and broken ones (open ones) stand for the ferric and ferrous cytochromes c3, respectively. (A) NH signals for Glu41 and Asp42 stand for triangles and squares, respectively. (B) NH signals for His67, Ala68, Asp71, and Phe76 stand for squares, triangles, circles, and diamonds, respectively. (C) NH signals for Leu9, Lys10, Lys45, and Cys46 stand for triangles, squares, diamonds, and circles, respectively.
Mentions: The pH titration curves of the amide protons of Glu41 and Asp42 in the oxidized and reduced states are plotted in Figure 4A. For the ferrous cyt c3, it is assumed that NH signal shifts did not influenced by the redox-equilibrium in the acidic pH region. These titration curves in each redox state are similar, that is, pKas of these titration curves are not dependent on the redox states. For the residues indicated large NH signal changes in both states, His67, Ala68, Asp71, and Phe76, pH titration curves are also plotted in Figure 4B. These residues also show the similar pH dependences. On the other hand, the titration curves for Leu9, Lys10, Lys45, and Cys46 amide proton signals plotted in Figure 4C revealed different pH dependences between the oxidized and reduced states.

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