Ferredoxin: Anything but a Toxin!(Because it’s essential to the plant)

AbstractAfter cloning cell colonies from the organism Landoltia Punctata, a strain of duckweed, DNA clone 105KL2.14 coded for a protein that matched up significantly with another protein involved in photosynthesis, known as ferredoxin. Plant [2Fe-2S] ferredoxins, iron-sulfur proteins found on the outside surface of the thylakoid membrane, serve primarily to transport electrons kept in the iron-sulfur complex from photosystem I (PSI) to either a lone thylakoid or to the ferredoxin-NADP+-Reductase enzyme (FNR), which reduces NADPH. Ferredoxins facilitate reduction of NADP+ to NADPH.

Stroma

Region of activity

How does it do it?Ferredoxin, with its [2Fe-2S] cluster, can transport electrons in it. Photosystem I collects the sunlight and uses light energy to transfer electrons to the ferredoxin. The iron in ferredoxin is reduced (gain of electron), and carries that electron in the cluster until it can hand it off to the FNR (where the iron is oxidized), which reduces NADP+ to NADPH with H+ from water. The NADPH is then used in the Calvin cycle in photosynthesis and then produces sugar.

BLASTp Results

Description: E-Valueferredoxin-1, chloroplastic [Solanum lycopersicum] (tomato) 2e-60 PREDICTED: ferredoxin-1, chloroplastic-like [Solanum tuberosum] (potato) 3e-60ferredoxin I [Solanum tuberosum] (potato) 3e-60PREDICTED: ferredoxin, chloroplastic-like [Glycine max] (soybean) 2e-59 Ferredoxin [Morus notabilis] (berry tree) 2e-59 2Fe-2S ferredoxin-like superfamily protein,A,ATFD2 [Theobroma cacao] (cacao) 3e-59

Alignment of our Protein to Parsley Ferredoxin

Differences in Amino AcidsThe differences in amino acids reside all the the outside corners of the protein, which makes sense because those aren’t very close to the iron-sulfur cluster, which is the main region of activity, making them not important to the function.

“Opening”

Ferredoxin has numerous identical amino acids found in many plants, as seen in the above diagram, which directly conveys its vital role in plants; conversely, organisms from other kingdoms did not show homology with ferredoxin, with the exception of bacteria. Some bacteria have bacteria-type Fe4S4 ferredoxin. Nonetheless, the matches in ferredoxin found in other plants implies those amino acids’ vital role in the function of ferredoxin, maybe to even stabilize the iron-sulfur cluster.

Homology of Ferredoxin

DiscussionWith Landoltia Punctata’s starch, another potential use for this particular strain is biofuel. It is important that photosynthesis, which produces the starch that is necessary for the production of biofuel, executes efficiently. Essentially, the more starch, the higher quality and more efficient the biofuel will be. Ferredoxin, the electron-transporter, is vital in the role of catalyzing photosynthesis and is therefore very important for this potential use as biofuel.

Highlighted in green are the binding sites that interact with FNR.

Binding sites to FNR

Two iron molecules, highlighted in orange, and two Sulfur molecules, highlighted in yellow, combine to make the [2Fe-2S] cluster; anchoring the the cluster to the protein are four cysteines, highlighted in gold.

Iron-Sulfur Cluster

Acidic Residues

MutationScientists (Benjamin A.Feinberg, Xiaoping Lo, Takeo Iwamoto, and John M.Tomich) created synthetic mutants of Clostridium pasteurianum ferredoxin. Several of these strains involved manipulating the role of cysteine within ferredoxin and, separately, adding multiple [2Fe-2S] clusters - all of which, overall, yielded mixed results. Nonetheless, replacing cysteines within the protein resulted in a fundamentally more unstable and less efficient ferredoxin.

ConclusionWith Ferredoxin’s essential role in photosynthesis, which is directly responsible for the massive amount of starch produced by duckweed, we can conduct further experiments to see if mutating ferredoxin produces more starch. Since altering any of the cysteine coordination sites will destabilize the protein, we can try mutating with other amino acids, to see if it will increase the efficiency of ferredoxin. If it were to increase the transfer of electrons in ferredoxin, photosynthesis could occur more frequently, which yields more starch and in turn, a higher quality of and a higher volume of biofuel.

Kangmin Lee & Robert MannifieldNorth Brunswick Township High School

Special Thanks to:Dr. O’Reilly - NBTHS WSSP AdvisorDr. Andrew Vershon - Course Instructor Mr. John Brick - Invaluable Lab AidDr. Janet Mead - Head of Laboratory Ms. Sue Coletta - Project CoordinatorAnd the rest of the WSSP Staff ...and to Gerlanda’s and Woody’s for sustenance