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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.


1H NMR spectra of axial ligand histidine C2 proton for wild-type and mutated ferric cytochromes c3 at 303 K. (A) wild type, (B) E41K, (C) Y43L20, (D) wild type, and (E) E41Q, respectively. (A–C) and (D, E) are recorded in 30 and 100 mM sodium phosphate buffers at p2H 7.0, respectively. Asterisks indicate the signals which could be assigned to His34.
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f9-2_45: 1H NMR spectra of axial ligand histidine C2 proton for wild-type and mutated ferric cytochromes c3 at 303 K. (A) wild type, (B) E41K, (C) Y43L20, (D) wild type, and (E) E41Q, respectively. (A–C) and (D, E) are recorded in 30 and 100 mM sodium phosphate buffers at p2H 7.0, respectively. Asterisks indicate the signals which could be assigned to His34.

Mentions: The Glu41 mutations showed large changes in the redox potentials at pH 7.0 (Tables 1 and 4). This is consistent with the previous report that the contributions of Glu41 to the electrostatic potential at heme irons of hemes 1 and 2 are relatively high34. On the other hand, the mutations of lysines 10, 15, 26, 57, 58, 60, 72, 94, 95, and 104 to methionine did not induce large changes in the macroscopic redox potentials18. In the case of the K101M mutant, the microscopic as well as the macroscopic redox potentials were the same as those of the wild type. The crystal structure of E41K showed a slight alteration of Tyr43 because of the hydrogen bond between Lys41 and Tyr43 (Fig. 8B). This alteration may change the strength of the π-π interaction between the aromatic ring of Tyr43 and the imidazole ring of His34, so that the microscopic redox potentials might increase. A strong coordination of His34 to Fe3+ would induce a slight polarization in the electron density of imidazole ring, which can be stabilized by π electron density of the Tyr43 ring through the π-π interaction. Thus, a redox potential would be lowered by the π-π interaction. A careful examination of the eight signals of the axial ligand histidine C2 protons in the 1H NMR spectra of the E41K, Y43L20, and E41Q mutants (Fig. 9) revealed significant shifts for the signal marked by asterisk in comparison with the wild type, supporting the idea that those mutations affect the coordination structure of a heme ligand. This signal could be assigned to the His34 because it is the closest histidine ligand to the Glu41 and Tyr43 residues. It should be noted that the crystal structures did not have enough resolution to show this kind of change in the coordination structure.


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
1H NMR spectra of axial ligand histidine C2 proton for wild-type and mutated ferric cytochromes c3 at 303 K. (A) wild type, (B) E41K, (C) Y43L20, (D) wild type, and (E) E41Q, respectively. (A–C) and (D, E) are recorded in 30 and 100 mM sodium phosphate buffers at p2H 7.0, respectively. Asterisks indicate the signals which could be assigned to His34.
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Related In: Results  -  Collection

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f9-2_45: 1H NMR spectra of axial ligand histidine C2 proton for wild-type and mutated ferric cytochromes c3 at 303 K. (A) wild type, (B) E41K, (C) Y43L20, (D) wild type, and (E) E41Q, respectively. (A–C) and (D, E) are recorded in 30 and 100 mM sodium phosphate buffers at p2H 7.0, respectively. Asterisks indicate the signals which could be assigned to His34.
Mentions: The Glu41 mutations showed large changes in the redox potentials at pH 7.0 (Tables 1 and 4). This is consistent with the previous report that the contributions of Glu41 to the electrostatic potential at heme irons of hemes 1 and 2 are relatively high34. On the other hand, the mutations of lysines 10, 15, 26, 57, 58, 60, 72, 94, 95, and 104 to methionine did not induce large changes in the macroscopic redox potentials18. In the case of the K101M mutant, the microscopic as well as the macroscopic redox potentials were the same as those of the wild type. The crystal structure of E41K showed a slight alteration of Tyr43 because of the hydrogen bond between Lys41 and Tyr43 (Fig. 8B). This alteration may change the strength of the π-π interaction between the aromatic ring of Tyr43 and the imidazole ring of His34, so that the microscopic redox potentials might increase. A strong coordination of His34 to Fe3+ would induce a slight polarization in the electron density of imidazole ring, which can be stabilized by π electron density of the Tyr43 ring through the π-π interaction. Thus, a redox potential would be lowered by the π-π interaction. A careful examination of the eight signals of the axial ligand histidine C2 protons in the 1H NMR spectra of the E41K, Y43L20, and E41Q mutants (Fig. 9) revealed significant shifts for the signal marked by asterisk in comparison with the wild type, supporting the idea that those mutations affect the coordination structure of a heme ligand. This signal could be assigned to the His34 because it is the closest histidine ligand to the Glu41 and Tyr43 residues. It should be noted that the crystal structures did not have enough resolution to show this kind of change in the coordination structure.

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.