A cell fractionation process was carried out in a bacterium with mutated cythocr

Question

A cell fractionation process was carried out in a bacterium with mutated cythocrome C. Based on the following table:

Cythocrome :

Fraction:

Activity Percentage:

Mutant and wt cythocrome C

GSH-FDH

PntAB

Cythocrome c2 (wt)

Cytoplasm

3.5%

95.3%

ND

Cytoplasmic Membrane

0.1%

1.9%

91%

Periplasm

96.4%

5.8%

9%

Outer Membrane

ND

ND

ND

Mutant cythocrome

Cytoplasm

0%

93%

100%

Cytoplasmic Membrane

100%

4.3%

ND

Periplasm

0%

2.7%

ND

Outer Membrane

ND

ND

ND

GSH-FDH = Glutathione-Dependent Formaldehyde Dehydrogenase

PntAB = nucleotide transhydrogenase

Question: Is there a problem with the mutant protein? What could be the cause, and how would you demonstrate it? - Hint: Pay close attention to the first row activity percentages of the mutant cythocrome C vs. the cythocrome C2 (wt). Please explain, with at least three paragraphs. Answer as thoroughly as possible and cite examples if required.

Cythocrome :

Fraction:

Activity Percentage:

Mutant and wt cythocrome C

GSH-FDH

PntAB

Cythocrome c2 (wt)

Cytoplasm

3.5%

95.3%

ND

Cytoplasmic Membrane

0.1%

1.9%

91%

Periplasm

96.4%

5.8%

9%

Outer Membrane

ND

ND

ND

Mutant cythocrome

Cytoplasm

0%

93%

100%

Cytoplasmic Membrane

100%

4.3%

ND

Periplasm

0%

2.7%

ND

Outer Membrane

ND

ND

ND

Explanation / Answer

Ans: Yes mutant protein that is cytochrome c having problem in export to periplasmic space hence in the periplasmic space fraction it is 0%, where as in wt it is 96.4%. cytochrome c has to be exported to periplasmic space. Due to this it cannot pump out electrons and free radicals accumulate in the cells, may ultimatlely lead to death. Periplasmic c-type cytochromes are initially synthesized as preapocytochrome forms carrying an N-terminal signal sequence that facilitates precursor translocation via the s-dependent preprotein translocase. The covalent coupling of the imported heme to the signal processed apocytochrome is generally thought to occur posttranslocationally in the more oxidizing periplasmic environment.

GSH-FDH: There is no mutation in this enzyme and helps in maintaining reduced glutathione pools so that glutathione can be oxidized to rmove free radicals from the cell.

PntAB has to be produced in cytoplasm and exported to cytoplasmic membrane, but in mutant strain it remained in cytoplasm hence it cannot do its function and cell ultimately die of free radical accumulation.

PntAB function: The enzyme couples hydride transfer of reducing equivalent between NADH and NADP+ to proton translocation across the membrane. Under most physiological conditions, the enzyme uses energy from the membrane proton gradient to produce high concentrations of NADPH. The resulting NADPH is used for biosynthesis as well as to remove reactive oxygen species such as to retain a reduced glutathione pool.

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