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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.


Chemical shift changes in the heme methyl signals of the E41K, E41Q, and K104M ferric cytochromes c3 in comparison with those of the wild type at p2H 7.0 and 303 K. E41K, E41Q, and K104M are compared with the wild type in 30, 100, and 100 mM sodium phosphate buffers, respectively. The heme methyl signals which are labeled alphabetically (A–J) from low to high magnetic field were used.
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f5-2_45: Chemical shift changes in the heme methyl signals of the E41K, E41Q, and K104M ferric cytochromes c3 in comparison with those of the wild type at p2H 7.0 and 303 K. E41K, E41Q, and K104M are compared with the wild type in 30, 100, and 100 mM sodium phosphate buffers, respectively. The heme methyl signals which are labeled alphabetically (A–J) from low to high magnetic field were used.

Mentions: 1D 1H NMR spectra were recorded in the oxidized state. To examine the effects of the mutations under the same condition, the spectrum of wild-type cyt c3 in the 100 mM NaPi buffer at p2H 7.0 was also recorded. The heme methyl signals in the low magnetic field (13–32 ppm), which are labeled alphabetically (A–J) from low to high magnetic field, were compared. For wild-type cyt c3, the increase of ionic strength from 30 mM to 100 mM NaPi did not induce any changes in these signals. The chemical shift assignments of the heme methyl signals of E41K were performed as will be mentioned later. For the E41Q and K104M mutants, only signals A–J were assigned in reference to those observed in E41K and wild type, respectively. The chemical shift differences between the wild type and mutants are summarized in Figure 5. The K101M mutation induced little change in the spectrum as previously reported18. The K104M mutation induced a relatively large change for signal E (heme 3), because Lys104 is close to heme 3. In the case of Glu41 mutants, signals for hemes 1 and 2 were mainly affected.


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
Chemical shift changes in the heme methyl signals of the E41K, E41Q, and K104M ferric cytochromes c3 in comparison with those of the wild type at p2H 7.0 and 303 K. E41K, E41Q, and K104M are compared with the wild type in 30, 100, and 100 mM sodium phosphate buffers, respectively. The heme methyl signals which are labeled alphabetically (A–J) from low to high magnetic field were used.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036644&req=5

f5-2_45: Chemical shift changes in the heme methyl signals of the E41K, E41Q, and K104M ferric cytochromes c3 in comparison with those of the wild type at p2H 7.0 and 303 K. E41K, E41Q, and K104M are compared with the wild type in 30, 100, and 100 mM sodium phosphate buffers, respectively. The heme methyl signals which are labeled alphabetically (A–J) from low to high magnetic field were used.
Mentions: 1D 1H NMR spectra were recorded in the oxidized state. To examine the effects of the mutations under the same condition, the spectrum of wild-type cyt c3 in the 100 mM NaPi buffer at p2H 7.0 was also recorded. The heme methyl signals in the low magnetic field (13–32 ppm), which are labeled alphabetically (A–J) from low to high magnetic field, were compared. For wild-type cyt c3, the increase of ionic strength from 30 mM to 100 mM NaPi did not induce any changes in these signals. The chemical shift assignments of the heme methyl signals of E41K were performed as will be mentioned later. For the E41Q and K104M mutants, only signals A–J were assigned in reference to those observed in E41K and wild type, respectively. The chemical shift differences between the wild type and mutants are summarized in Figure 5. The K101M mutation induced little change in the spectrum as previously reported18. The K104M mutation induced a relatively large change for signal E (heme 3), because Lys104 is close to heme 3. In the case of Glu41 mutants, signals for hemes 1 and 2 were mainly affected.

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.