Life System and Environmental & Evolutionary Biology II

Lab 10:  Enzymes and Photosynthesis

Kevin Griffin

Part A:

Overview: Photosynthesis is the process by which green plants use sunlight to make glucose from carbon dioxide and water. The glucose made by plants can then be used by plants and animals as a source of energy by releasing the energy contained in the bonds of glucose and converting it to ATP in cellular respiration. Respiration also produces waste products including carbon dioxide and water, which are the same substances that served as the raw materials for photosynthesis. In water, carbon dioxide dissolves to form a weak acid and as a result, an acid-base indicator such as bromothymol blue can be used to indicate the presence of carbon dioxide. In this laboratory investigation, you will use bromthymol blue to explore the relationship between photosynthesis and respiration.

Problem: What is the relationship between the processes of photosynthesis and respiration? What gases are exchanged?

Procedure: WORK IN TEAMS OF TWO OR THREE!

1. Using a water bottle, wash a philodendron leaf and then gently pat it dry.  Using a razor blade, remove the leaf from the plant, slice it length-wise along the midrib, roll it into a small cylinder and insert each half of the leaf into a glass scintillation  vial. Be sure the bottom surface of the leaf is NOT against the glass, but points in towards the center of the vial.  Place a cap on the vials and label them (keep the label small so you do not block the light).  Now completely wrap one of the two vials with aluminum  foil.  Place the two vials under the light for 20 min.

2.  Your TA will demonstrate the reaction with the bromthymol blue solution by gently blowing air into a small volume of the solution until there is a change in the appearance of the bromethymol blue solution.  Once the solution has changed color your TA will carefully transfer half of the solution into each of the vials you set up in part one.  It is critical that the solution be added with out disturbing or mixing the air more than is necessary.  Replace the cap on the vial, make sure the "dark" vial is still completely covered with aluminum foil and place both vials back under the light.  Record your observations, including what happens to the dye when you blow into it and what effect the light and dark treatments have on the dye (record the color of the solution every 10 mins. for the remainder of the lab).  Be sure to gently shake the vial each time you check the color, but avoid getting the dye on the leaf directly.

Caution: Bromthymol blue is a dye and can stain your hands and clothing.

Analysis:

  1. What was the color of the bromthymol blue solution before your TA exhaled into it? After they blew into it? Why did it change color?

  2. Would it be possible to change the color back with your breath?

  3. Why did we use bromthymol blue in this experiment?

  4. Why was a philodendron leaf placed in both flasks?

  5. What would happen if you used twice as many leaves?

  6. What differences did you observe between the light and the dark treated leaves? Why did this occur?

  7. How do your results demonstrate the products and reactants necessary for photosynthesis and respiration?

  8. Where did the energy needed for the conversion of bromethymol blue come from?

  9. Was entropy a part of the this experiment?

Part B: How do Proteins Help Chlorophyll Carry Out Photosynthesis?

In Photosynthesis, photons of light are absorbed by chlorophyll molecules, causing them to donate a high-energy electron that is put to work making NADPH and pumping protons to produce ATP. In this section of the lab you will examine recent evidence that proteins embedded in the thylakoid membranes within the chloroplasts of photosynthetic organisms are acting as an antenna to facilitate light capture.

This section of the lab can be found on the textbook web page by accessing this link: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/.

From here select "Online Labs" from the menu list on the left of the screen. Then select: "Virtual Lab 5 - How Do Proteins Help Chlorophyll Carry Out Photosynthesis?"

From here you can check out the various resources and background information. Ultimately you will want to "Run a virtual Experiment…" by selecting the appropriate button. Simply follow along the instructions.

For your lab report please give the hypothesis tested and major conclusions. Also answer the 3 additional questions posed:

  1. Would PSI function the same in the mutant as it does in the wildtype?

  2. Would your experiment give the same results if you substitute a different amino acid for the histidine?

  3. Do your results support or reject your hypothesis?

Part C:

In this weeks lectures we explored the relationships between living things and energy transfer.  In this lab we will more specifically consider the relationships between energy and mass transfer in photosynthetic carbon gain.  For the next part of this lab we will utilize the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB).  Go to the Expasy Home Page and search for "Ribulose-bisphosphate carboxylase".  From the description answer the following questions:

  1. What are two other names for this enzyme?

  2. What is the reaction catalyzed by this enzyme?

  3. How many carbons are in the product of this reaction? (If you're having trouble, check the Fischer diagram of the linked web page in the reaction.)

  4. Why do the alternative names for the enzyme include Ribulose bisphosphate carboxylase/oxygenase? (If you're having trouble, check the "Reaction catalyzed" in the BRENDA entry link )

  5. Now view the Biochemical Pathways map (number U10), and from it answer these questions:

    1. What are the immediate inputs to this reaction?

    2. What are the immediate products of this reaction?

    3. What cofactors are required?

    4. What is the ultimate product of oxygenation and how many carbon atoms does it contain?

    5. What enzyme regulates the step immediately prior to the one catalyzed by Rubisco?

    6. What enzyme catalyses the reaction immediately following the one catalyzed by Rubisco?

    7. Is energy (ATP) directly required for the reaction catalyzed by Rubisco?

    8. Is energy (ATP) indirectly required for the reaction catalyzed by Rubsico?  If so how and where would it come from?
       

Part D:

Enzyme kinetics can be mathmatically modeled allowing us to do thought experiments without getting our hands dirty!  The simplest approach is known as a Michaelis-Menton equation describing the activity of an enzyme in response to substrate availability.  The basic response is illustrated below.

Michaelis-Menton Equation

v = velocity

V= maximum velocity (Vmax)

a = [S]

Km = Michaelis-Menten Constant (curvature)
 
 

At 25░C Rubisco Kinetics in Soybean:

Short-term photosynthetic response to CO2

Vcmax = 100 Ámol m-2 s-1

Kc = 441 ppm

Vomax = 22.8 Ámol m-2 s-1

Ko = 248000 ppm

Vomax/Vcmax = 0.22

This is not quite as crazy as it seems, remember the atmosphere is 210,000 ppm O2 and only 360 ppm CO2!


 
 

Mathematical Models of Photosynthesis:

Since Rubisco has both an oxygenation and carboxylation reaction, we can not describe the overall reaction with a simple Machaelis-Menten equation without taking into account the competitive interaction of the two substrates.

Net CO2 assimilation (A - photosynthesis or carbon fixation) is the result of the rate of carboxylation (Vc) minus phtosrespiration (oxygenation) and mitochondrial respiration. In photorespiration, one CO2 is produced per two oxygenation reactions (Vo). So:

A = Vc - 0.5Vo - Mitochondrial Respiration (1)

An effective Michaelis-Menten constant for the carboxylation reaction that takes into account the competitive inhibition of by O2 is:

Km = Kc(1+O/Ko) (2)

The Rate of carboxylation can then be described as:

(3)

It is important to point out that the above equation describes only the CO2 limited portion of the CO2 response curve of photosynthesis. In reality other factors often become limiting long before the Rubisco reaction is saturated (what might some of thoses be?). Also as we point out above we would need to take into account respiration and the oxygenation reaction to have an accurate model of photosynthesis. These models exist and are regularly used by plant physiologist. Today we will stop here and consider the implications of these reactions rather than the equations describing them.
 

ALWAYS LABEL THE X AND Y-AXES OF YOUR PLOTS!

  1. Use Microsoft Excel to recreate the Rubisco graph above, plotting the response curves for the oxygenation and carboxylation reactions.

    1. Now experiment with changing the values for Kc and Ko and describe what happens.
    2. Make a new plot of the carboxylation reaction with Kc set to 150, 350 and 750 Ábar and interpret your result. Which is the more "efficient" plant?
    3. Make a new plot of the carboxylation reaction with V= 75, 125 and 225 Ámol m-2 s-1 and interpret your result (set Kc back to 400) . Which is the more "efficient" plant?
  2. Now calculate the Km using equation 2 above.

    1. Make a new plot of photosynthesis using Km (set V back to 100). On this same plot ad the curve for Kc from part A.
    2. Interpret these results as compared to the first plot of Kc and Ko.
  3. Human caused increases in atmospheric CO2 are predicted to double the CO2 by the middle to end of the next century (from 360 to 720 ppm CO2). Based on your results above, what do expect to happen to photosynthesis ?

  4. Whats the point? So what, we can make mathmatical models of enzyme reactions driving photosynthesis. Why do you think we should care about all of this? (If you are having trouble I suggest you take a deep breath!)