ANSWER KEY 

Exam on OXPHOS and Photosynthesis	
David Nelson's lectures (30 pts)				Oct. 30, 1996

1a) (2 pts) Describe four similarities between the bc1 complex (Complex III) and the 
cytochrome b6/cytochrome f complex of photosynthesis.  Include the carriers that bring 
electrons to and from the complexes.  

	a. plastoquinone is similar to ubiquinone, both deliver electrons to the complex
	b. cytochrome f is similar to cytochrome c1
	c. both complexes have the Rieske iron sulfur protein
	d. plastocyanin carries electrons from the complex, like cytochrome c

	Alternative answers:

	e. both have b cytochromes
	f. both pump protons as electrons are transferred

1b) (1 pt) What is the major difference between these two complexes.

The b6f complex does not operate a Q cycle.

2) (2 pts) You have isolated a respiration defective mutant in yeast and you want to 
characterize where the defect is.  You isolate intact mitochondria from the yeast.  In an 
oxygen electrode measurement with ADP and Pi present, beta-hydroxybutyrate does not 
cause any consumption of oxygen.  Succinate does cause oxygen consumption.  This is 
blocked by stigmatellin and Antimycin A, but rotenone has no effect.  Which complex is 
affected by the mutation.

Complex I is affected.

3) (3 pts) Mitochondria are said to be coupled when electron flow to oxygen depends on 
ADP and Pi.  Even if a source of electrons is present (succinate, or beta-hydroxybutyrate) 
no oxygen is consumed without substrates for ATP synthase.  Why can't the electrons 
flow to reduce oxygen under these conditions?  

The ATP sysnthase is like a valve controlling the proton gradient movement across the 
inner membrane.  When no substrate is present the valve is closed and maximum pressure 
builds up (the gradient is maximized).  When the substrate is present then the valve is 
opened and protons can flow.  No electrons can flow to oxygen with the gradient 
maximized, because there is not enough energy released by electron transport to pump 
more protons against the gradient.  

4) (2 pts) This paragraph was taken from OMIM (Online Mendelian Inheritance in Man).  

Riggs et al. (1984) described 2 sibs with deficiency of complex II. A 7-year-old boy and 
his 9-year-old sister had progressive encephalomyopathy with dementia, myoclonic 
seizures, and short stature. A muscle biopsy showed mitochondrial aggregates and 
excessive lipid droplets in muscle fibers. In muscle mitochondria, the activity of succinate 
cytochrome c reductase was deficient. All succinate dehydrogenase activity was normal. 
The activities of NADH-cytochrome c reductase and cytochrome oxidase were also normal. 
The defect was thought to lie somewhere between succinate dehydrogenase and coenzyme 
Q(10) in complex II. 

Please note that the ability to transfer electrons to complex III (succinate cytochrome c 
reductase) is distinct from the ability to function in the TCA cycle and oxidize succinate 
(succinate dehydrogenase).  Suggest two possible defects that might exist in complex II.  

There is probably a defect in the electron transfer path from FADH2 to the ubiquinone 
binding site that blocks electron movement, or the ubiquinone binding is defective.

5) (3 pts) What are the three main features of the binding change mechanism of ATP 
synthesis?

1. Energy released by proton flux through the ATP synthase is required to release tightly 
bound ATP from the enzyme.  It is not required to form the ATP.

2. All three catalytic subunits are in different conformations.

3. The rotation of the F1 ball on the gamma subunit is responsible for interconversion of 
the three catalytic sites between the open, loose and tight configurations.  This rotation is 
coupled to proton movements.

6) (3 pts) Describe Richard Cross' experiment that showed F1 rotates relative to the gamma 
subunit.

A mutation in the beta subunit introduces a cys residue that can crosslink with cys87 of 
gamma.  A disulfide locks the enzyme and prevents rotation.  To show rotation really 
occurs, the disulfide linked enzyme is dissociated and reconstituted with additional beta 
subunits that have an epitope tag.  Now only the original beta subunit that was cross-linked 
to beta is not tagged.  The enzyme is reconstituted with F0 and reduced.  Substrate is added 
and the enzyme allowed to rotate.  The disulfide is reformed and the complex is examined 
on a Western blot looking for epitope tagged beta cross-linked to gamma.  If this is found 
then the subunits rotated, and that is what was seen.

7) (2 pts) Propionigenium modestum has an F1F0 ATPase that works with Na+ and at a 
low sodium concentration it will transport H+.  If you could change one subunit of the E. 
coli enzyme, by swapping genes, which subunit would you change to try and switch the E. 
coli enzyme to a Na+ ATPase?

The proton channel is dependent upon the small c subunit, the DCCD binding protein.  
This is the subunit to change.  The a subunit is also involved in the channel for protons, so 
either a or c would be acceptable answers.  F0 will get half credit.

8) (2 pts) How did Efriam Racker and Walther Stoeckenius use a reconstitution experiment 
to verify Peter Mitchell's chemiosmotic hypothesis?

ATP synthase was reconstituted in phosholipid vesicles with bacteriorhodopsin, a light 
driven proton pump.  When light shines on the vesicles, the interior becomes acid and the 
ATP synthase makes ATP.  For this experiment ot work both the bacteriorhodopsin and the 
ATP synthase have to be oriented correctly in the membrane.

9) (3 pts) Describe the regulation of the rubisco enzyme, and tell how it is affected by light.

Rubisco is activated by alkaline pH, which is created by photosynthesis proton pumping 
into the thylakoid lumen.  This aids in the required carbamylation of lys 201.

Rubisco activase is also activated at alkaline pH

Mg is pumped out of the lumen to the stroma in the light and Mg is required for rubisco 
activity.

10 (2 pts) What is photorespiration?  Give one example of a mechanism some plants use to  
limit the photorespiration pathway.

Rubisco can use oxygen instead of CO2.  This produces phosphoglycolate that is oxidized 
to CO2.  This is photorespiration.  

C4 plants convert CO2 to a four carbon acid and import it away from the plant surface to 
the interior where O2 concentration is lower.  

Crassulacean acid metabolism does a similar thing, only the acid is formed at night and 
broken down during the day to free the CO2 when light is present.  This also reduces the 
oxygen concentration.  

11a) (1 pt) Purple bacteria photosynthesize without a photosystem II.  They do not split 
water to make oxygen.  What is the fate of the excited electron in the purple bacterial 
reaction center?  

It returns to the place it started, the bacterial light reaction center, after cycling through the 
bc1 complex.  

11b) (1 pt) What process in the Z-scheme of photosynthesis is similar to purple bacterial 
photosynthesis.

cyclic photophosphorylation

12) (3 pts) Describe the flow of an electron through photosystem I.  Start with P700 and 
end with NADPH.

P700 is excited, the elctron moves to Ao (a chlorophyll) then to A1 (a quinone).  The 
electron moves to iron sulfur centers FeSx FeSA FeSB then to ferredoxin, a soluble 
electron carrier that takes the electron to ferredoxin NADP+ reductase where NADPH is 
formed.