Research On The Prairies – All You Wanted To Know About Chlorite Dismutase

Participating in an REU program was definitely an experience I was looking forward to as a chemistry major.  After all, what better way to glimpse life as a graduate student conducting new and exciting research at an advanced level than to have the opportunity to work in a research group alongside graduate students conducting new and exciting research at an advanced level?  I was a little concerned during the application process since the chance to tour the lab and meet in person with the professor is definitely worth doing if at all possible, and while the program I was most interested in happened to be just across the river from my home institution, my current institution for the semester (the National University of Ireland in Galway) happened to be just across the ocean.  Luckily, Skype works quite well for transcending geographical and temporal hurdles, and, as can probably be surmised, I eventually found myself spending quite a lot of time on the Fargo side of the Red River.

The Rodgers group is one that sort of creates a niche of its own.  The molecule heme is a running theme in all of the ongoing projects and is examined using multiple spectroscopic techniques including UV-Visible and resonance Raman, the latter of which is laser-based.  Heme is an aromatic structure that contains an iron center surrounded by a porphyrin ring with characterizing substituents.  It is a prevalent cofactor and plays a role in a broad spectrum of proteins.  This includes hemoglobin, which is responsible for oxygen transport (among other functions) and is what lends blood its red color.The metal component of heme coupled with its role with so many different proteins and biological processes results in the research falling under not solely inorganic or biochemistry but an amalgamation of the two dubbed bioinorganic chemistry.

The all-important cofactor, heme b

The same type of heme found in hemoglobin (heme b) is also found in my focus for the summer, a very unique group of bacterial and archael enzymes called chlorite dismutases (Cld).  What sets this superfamily of proteins apart is their ability to decompose chlorite (ClO2) into chloride (Cl) and oxygen (O2).  The capacity to produce O2 is recognized in only a few characterized enzymes.  Another more familiar example would be photosynthesis with photosystem II.Chlorite dismutases also become very significant in their potential for bioremediation.  ClO2 and other chloro-oxyanions like perchlorate (ClO4), chlorate (ClO3), and hypochlorite (ClO) present quite the environmental and health hazards due to their ubiquitous use as herbicides, explosives, and industrial bleaching agents.  In fact, the EPA categorizes ClO2 as one of the top ten pollutants in drinking water, and the health hazard it presents generally targets children in the forms of neonatal jaundice and childhood anemia.ClO2 can also disproportionate in acidic conditions into ClO, otherwise known as household bleach, and cause some pretty nasty chemistry.  Consequently, an enzyme that not only produces oxygen but detoxifies the environment certainly merits a look.

The mechanism of Cld is currently under investigation, and the goal of the study is to fully detail the relationship between the function of the enzyme and the structure of the enzyme.  Below is a proposed mechanism for how it goes about the decomposition.4  ClO2 enters the active site of the protein and attacks the iron center.  An oxygen is left behind as the molecule releases as ClO.  ClO reorients and forms a peroxy or oxygen-oxygen bond before coming off as O2 and Cl.   While there is evidence for the initial steps of the mechanism, full confirmation has yet to be achieved.  This is partly because, as of yet, none of the intermediates hang around long enough to be isolated.

  In order to conduct experiments on our proteins, we first needed to have them.  This involved a week-long process of culturing, inducing protein expression, harvesting, freezing (via liquid nitrogen, always fun), ultrasonication, and purification via column chromatography that, ideally, would end with milligrams of new, shiny (not literally) protein to work with.  Of course, as my graduate student mentor Zach Geeraerts and I found out, protein preps can sometimes be generously described as problematic.  While it may have delayed our timeline a bit, it was always a good exercise in reasoning what the problem was, testing and confirming that it was indeed the problem, and altering the procedure to reflect the new information.  Considering that research is often breaking new ground in its field, being able to nail down that process is invaluable.

A column filled with resin used for purifying protein.  Much protein was sacrificed to appease the column deities. 

 We worked with two strains of Cld.  One was a soil bacteria by the name of Dechloromonas aromatica and the other was Klebsiella pneumoniae.  The experiments, once they got under way, dealt with activity measurements and nitric oxide adducts.  The former involves measuring how much O2 is produced by the enzyme when introduced to a ClO2 solution.  The probe we use has a ruthenium complex in its tip that, when excited by an LED, luminesces.  When O2 is present, that luminescence signal undergoes Stern-Volmer quenching, and the signal from the probe decreases.  The more O2 produced, the lower the signal, and the higher the enzyme’s activity.

On the left is the activity assay for the Klebsiella.  The right is for Dechloromonas.  From the steepness of the curve, one can see that Dechloromonas has a higher activity.

The nitric oxide (NO) adducts involved coordinating or bonding NO to the iron center in order to emulate the chemistry of potential intermediates.  Through UV-Visible spectroscopy, we were able to confirm that we had made both the {FeNO}6 form its reduced form {FeNO}7.  The next step would be to use resonance Raman spectroscopy to explore the positioning of the NO in the active site, where the electron density is, and what the hydrogen bonding environment is like.  As I will be continuing in the Rodgers lab, I will have the pleasure of seeing how these experiments play out during the semester.


1.  Streit, B.; Blanc, B.; Lukat-Rodgers, G.S.; Rodgers, K.R.; DuBois, J.L. J. Am. Chem. Soc. 2010, 16, 5711-5724.

2.  Hofbauer, S.; Schaffner, I.; Furtmüller, P.G.; Oblinger, C. Biotechnol. J. 2014, 9, 461-473.

3.  Streit, B.; DuBois, J.L. Biochemistry. 2008, 19, 5271-5280.

4.  Lee, A.Q.; Streit, B.R.; Zdilla, M.J.; Abu-Omar, M.M.; DuBois, J.L. Proc. Natl. Acad. Sci. USA 2008, 105, 15654-15659.


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