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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.


Comparison of the crystal structures of wild-type, E41K, and E41Q cytochromes c3. The four heme architecture (A) and the region between hemes 1 and 2 (B) of the wild type, E41K, and E41Q under the best fitting for the Cα of K3–C105 residues. The structure models are color-coded red, green, and purple for the wild type, E41K, and E41Q, respectively. These figures were drawn with MOLMOL38.
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f8-2_45: Comparison of the crystal structures of wild-type, E41K, and E41Q cytochromes c3. The four heme architecture (A) and the region between hemes 1 and 2 (B) of the wild type, E41K, and E41Q under the best fitting for the Cα of K3–C105 residues. The structure models are color-coded red, green, and purple for the wild type, E41K, and E41Q, respectively. These figures were drawn with MOLMOL38.

Mentions: The refinements of the crystal structures for the oxidized E41K (PDB code 1WR5) and E41Q (PDB code 2FFN) cyt c3 have been completed at 1.40 and 1.80 Å resolution, respectively. The crystallographic R- (free R-) factors of E41K and E41Q are 13.83 (21.83) and 17.04 (21.66) %, respectively. 17 ethanol molecules were identified in the E41K structure. The overall structures of E41K and E41Q cyt c3 were almost identical to that of the wild type20, as shown in Figure 8. The root mean square deviations (rmsd) of E41K and E41Q with respect to the wild type for all identical atoms in the residue range from K3 to C105 and four hemes were 0.58 and 0.53 Å, respectively. In the crystal structure of E41K, a hydrogen bond was formed between Oη atom of Tyr43 and Nζ atom of Lys41 (3.30 Å). The conformation of Gln41 in E41Q was similar to Glu41 in the wild type (Oε1(Gln41) − Oη(Tyr43) = 2.82 Å, Oε1(Glu41) − Oη(Tyr43)=2.75 Å, Fig. 8B).


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
Comparison of the crystal structures of wild-type, E41K, and E41Q cytochromes c3. The four heme architecture (A) and the region between hemes 1 and 2 (B) of the wild type, E41K, and E41Q under the best fitting for the Cα of K3–C105 residues. The structure models are color-coded red, green, and purple for the wild type, E41K, and E41Q, respectively. These figures were drawn with MOLMOL38.
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Related In: Results  -  Collection

Show All Figures
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f8-2_45: Comparison of the crystal structures of wild-type, E41K, and E41Q cytochromes c3. The four heme architecture (A) and the region between hemes 1 and 2 (B) of the wild type, E41K, and E41Q under the best fitting for the Cα of K3–C105 residues. The structure models are color-coded red, green, and purple for the wild type, E41K, and E41Q, respectively. These figures were drawn with MOLMOL38.
Mentions: The refinements of the crystal structures for the oxidized E41K (PDB code 1WR5) and E41Q (PDB code 2FFN) cyt c3 have been completed at 1.40 and 1.80 Å resolution, respectively. The crystallographic R- (free R-) factors of E41K and E41Q are 13.83 (21.83) and 17.04 (21.66) %, respectively. 17 ethanol molecules were identified in the E41K structure. The overall structures of E41K and E41Q cyt c3 were almost identical to that of the wild type20, as shown in Figure 8. The root mean square deviations (rmsd) of E41K and E41Q with respect to the wild type for all identical atoms in the residue range from K3 to C105 and four hemes were 0.58 and 0.53 Å, respectively. In the crystal structure of E41K, a hydrogen bond was formed between Oη atom of Tyr43 and Nζ atom of Lys41 (3.30 Å). The conformation of Gln41 in E41Q was similar to Glu41 in the wild type (Oε1(Gln41) − Oη(Tyr43) = 2.82 Å, Oε1(Glu41) − Oη(Tyr43)=2.75 Å, Fig. 8B).

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.