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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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1H NMR spectra of wild-type (A) and E41K (B) ferric cytochromes c3 at a variety of temperatures. Only fingerprint regions are presented. (C) The temperature dependences of line-widths of the heme methyl signals for the wild type and E41K at 600 MHz. Signal B for heme 1, signal C for heme 2, signal E for heme 3, and signal A for heme 4 are represented by diamonds, squares, triangles, and circles, respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
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f6-2_45: 1H NMR spectra of wild-type (A) and E41K (B) ferric cytochromes c3 at a variety of temperatures. Only fingerprint regions are presented. (C) The temperature dependences of line-widths of the heme methyl signals for the wild type and E41K at 600 MHz. Signal B for heme 1, signal C for heme 2, signal E for heme 3, and signal A for heme 4 are represented by diamonds, squares, triangles, and circles, respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.

Mentions: Temperature dependence of 1D 1H NMR spectra in the oxidized state was examined in the range 283–313 K. Generally, line-widths of heme methyl signals gradually broaden according to the decrease of temperature. For the wild-type cyt c3, the line-width of each heme methyl signal showed the similar tendency in this temperature range (Fig. 6A). The signals for the E41Q, K101M, and K104M mutants revealed similar behavior. For the E41K mutant, however, an obvious change of line-width in the low temperature range was observed (Fig. 6B). Signals B due to heme 1, C due to heme 2, E due to heme 3, and A due to heme 4 of the wild-type and E41K cyt c3 were fitted to lorentzian using Felix version 2000.1 (Accelrys Software Inc.). Obtained line-widths were plotted as a function of temperature in Figure 6C. The line-width of signal B for E41K cyt c3 significantly broadened in the low temperature range, which revealed that E41K mutation slowed the exchange rate in a conformational equilibrium around heme 1.


Roles of charged residues in pH-dependent redox properties of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki F
1H NMR spectra of wild-type (A) and E41K (B) ferric cytochromes c3 at a variety of temperatures. Only fingerprint regions are presented. (C) The temperature dependences of line-widths of the heme methyl signals for the wild type and E41K at 600 MHz. Signal B for heme 1, signal C for heme 2, signal E for heme 3, and signal A for heme 4 are represented by diamonds, squares, triangles, and circles, respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036644&req=5

f6-2_45: 1H NMR spectra of wild-type (A) and E41K (B) ferric cytochromes c3 at a variety of temperatures. Only fingerprint regions are presented. (C) The temperature dependences of line-widths of the heme methyl signals for the wild type and E41K at 600 MHz. Signal B for heme 1, signal C for heme 2, signal E for heme 3, and signal A for heme 4 are represented by diamonds, squares, triangles, and circles, respectively. Solid lines (closed symbols) and broken ones (open ones) stand for the wild type and E41K, respectively.
Mentions: Temperature dependence of 1D 1H NMR spectra in the oxidized state was examined in the range 283–313 K. Generally, line-widths of heme methyl signals gradually broaden according to the decrease of temperature. For the wild-type cyt c3, the line-width of each heme methyl signal showed the similar tendency in this temperature range (Fig. 6A). The signals for the E41Q, K101M, and K104M mutants revealed similar behavior. For the E41K mutant, however, an obvious change of line-width in the low temperature range was observed (Fig. 6B). Signals B due to heme 1, C due to heme 2, E due to heme 3, and A due to heme 4 of the wild-type and E41K cyt c3 were fitted to lorentzian using Felix version 2000.1 (Accelrys Software Inc.). Obtained line-widths were plotted as a function of temperature in Figure 6C. The line-width of signal B for E41K cyt c3 significantly broadened in the low temperature range, which revealed that E41K mutation slowed the exchange rate in a conformational equilibrium around heme 1.

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