Lab 5: Light Driven Proton Pumping in Spinach Chloroplasts

Question 1

This reaction, is when chlorophyll molecules are energized by light, water is hydrolyzed and O2 gas is produced.

Answer 1

hill reaction

Question 2

reaction for the hill reaction

Answer 2

12 H20 + 12 NADP + 18ADP + 18Pi —-> 6O2 + 18ATP + 12 NADPH

Question 3

Where does the hill reactions occur?

Answer 3

in the thylakoid membrane

Question 4

the _____ consumes NADPH and ATP made by the light reactions in order to “fix” CO2 into glucose.

Answer 4

the calvin cycle.

Question 5

reaction for the calvin cycle

Answer 5

12 NADPH + 18 ATP + 12 CO2 —-> Glucose + 18 ADP + 18 Pi + 12 NADP + 6 H2O

Question 6

during the light reactions, H+ ions are pumped from the _____ to the ______

Answer 6

pumped from the stroma into the thylakoid space.

Question 7

When the light reactions are occurring, it is expected that the thylakoid lumen is _____ while the stroma is ____

Answer 7

lumen is acidic and the stroma is basic.

Question 8

If you were to cover a plant up with a black garbage bag, over a period of time, what will happen to the pH of the stroma in the chloroplasts?

Answer 8

you would lose the proton gradient because protons cannot be pumped into the lumen as there are no more light reactions taking place. Thus, protons will remain in the stroma and you can expect a pH decrease

Question 9

two reasons as to why the PH of the stroma decreases when you inhibit the light reactiosn

Answer 9

1) proton gradient is lost because no light reactions are happening
2) the proton gradient that was there is diminishing because protons are flowing back to the stroma through ATP synthase (creates ATP at the same time)

Question 10

the ____ force drives protons through ATP synthase in the thylakoid membrane from the lumen to the stroma

Answer 10

proton motive force

Question 11

on what face of the thylakoid membrane is ATP synthesized?

Answer 11

in the stroma

Question 12

how many protons need to pass through ATP synthase to generate one ATP in the chloroplast?

Answer 12

3 protons.

Question 13

prior to starting the experiment, the leaves were left in the cold and dark. Why?

Answer 13

to reduce starch content. By keeping it cool and in the dark, the light and dark reactions of photosynthesis are kept to a minimum, and thus glucose cannot be made and stored as starch

Question 14

Why did we suspend the chloroplasts in an NaCl solution after centrifuging the spinach?

Answer 14

to swell the chloroplasts and make the outermembrane LEAKY so that pH of the stroma could be detected in an external medium

Question 15

the concentration of chlorophyl content in spinach wants to be determined. The absorbance of the chlorophyl solution is 0.28, which was multiplied by constant 5.6 to get 1.568mg/ml. We want a 15 ml solution of 0.5mg/mL chloroplasts. How much of the original chloroplast solution do we need?

Answer 15

use c1v1=c2v2. answer should be 4.783 mL of chloroplast solution in 10.2 ml of H2O.

Question 16

CCCP is a proton ionophore. What would happen if you added this to a chloroplast solution?

Answer 16

you would see a pH drop in the stroma, even IF THE LIGHTS ARE ON.

• light reactions would pump H+ into the lumen, but the proton gradient would be lost because protons are allowed to flow back into the stroma by the ionophore channel created by CCCP
• ATPase would not be powered because there is no PMF, therefore no ATP generated.

Question 17

How is oxygen evolution affected by proton ionophores?

Answer 17

O2 is still being evolved, but the proton gradient is uncoupled and allows for H+ to flow back into the stroma after being pumped.

Question 18

How does CCCP act as a proton ionophore?

Answer 18

delocalized the H+ charge by containing aromatic rings, this interaction allows for transport of H+ back into the stroma from the lumen.

Question 19

Main difference between PMF in Mitochondria and PMF in chloroplasts

Answer 19

In mito: PMF is created by using the free energy of redox reactions involving NADH and FADH2 tranferring their electrons to complex I, III or IV in order to pump protons across the membrane into the inner membrane space. The pumping of protons results in a change in voltage and change in conc, resulting in a change in pH, which can be quantified as the pmf. ATP synthesis is overall conducted via oxidative phosphorylation

in Chloroplast: DOES NOT use free energy from redox reactions to facilitate proton pumping. INSTEAD, it uses light energy to photolyze water and transfer its electrons through various photosystems and proton pumps, allowing protons to move to the lumen of the thylakoid before the electrons ultimately reduce NADP+, producing O2 as a biproduct. Like the mito, the H+ ions can move through ATP synthase to create ATP, a process called chemiosmosis.