Dean Wheeler, Chemical Engineering

Purpose

As described in the proposal submitted last year for this project, there is a great need to further understand the dynamics and processes of the nitrogenase system. Using this chemical system a limited number of microorganisms are able to break down atmospheric nitrogen so it may be used by other organisms. The heart of the matter-where the reduction of nitrogen takes place-is a small cluster of atoms located inside the dinitrogenase protein. This cluster is known as the iron-molybdenum cofactor or FeMoco.

The cofactor is currently thought to exist as a spheroidal-shaped combination of one molybdenum, six iron, and six sulfur atoms, as well as other variable adducts. Researchers are not certain exactly how the reduction reaction is carried out, but are looking toward the cofactor’s unusual structure for answers. Most contemporary research involves detailed structural studies using powerful analytical instruments and computers, which allow researchers to learn about the cofactor while it is buried inside the protein structure.

On the other hand, my proposal was to first extract the cofactor from its protein complement, complex it with various molecular species, and then purify and characterize the resulting products. By isolating the cofactor and testing its complexation properties, it was hoped that further information on its structure and properties could be deduced.

Procedure

Nitrogenase is highly sensitive to oxygen. Even extremely small amounts of oxygen will inactivate the nitrogenase components. Therefore, all experiments were carried out under an inert argon atmosphere. This was accomplished in special anaerobic flasks and in a glove box. All solutions contained a small amount of sodium dithionite, which would neutralize any residual oxygen. The first step in this investigation was to isolate the cofactor. Although there is a published procedure for accomplishing this, it was found that refinements to the protocol needed to be made. It was important to optimize the procedure because of the difficulty in obtaining even milligram quantities of cofactor. First, previously obtained frozen samples of nitrogenase proteins from the bacteria azotobacter vinelandii were thawed. Then, the solution was carefully acidified to pH 2.5, followed by neutralization to pH 7. This step unfolds the proteins, leaving the cofactor intact. The precipitated protein was separated from most contaminants using centrifugation. Next, the cofactor was extracted from the protein by the organic solvent N-methyl formamide (NMF) at pH 7.75. After centrifugation, the supernatant fluid, which contains the FeMoco clusters, was concentrated by evaporation under vacuum. Finally, the concentrated cofactor solution is frozen in liquid nitrogen for preservation.

In the next phase of study, the cofactor was further purified. Gel permeation liquid chromatography was used. Using the NMF solvent and Sephadex G-25 microbeads, 1 em diameter glass columns were poured. After passing through the column, the cofactor solution resolved into two principle peaks, as detected by a continuous flow cuvette installed in a spectrophotometer. The first peak was the healthy cofactor, while the second peak was likely cofactor fragments and other impurities.

Alternately, the cofactor was reacted with either excess tungstate or excess adenosine triphosphate (ATP) in an effort to complex it. The resulting solutions were passed down the colunm as described above. This also seemed to yield successfully purified cofactor fractions.

The greatest obstacle to the research was trying to characterize the cofactor fractions. The BYU Chemistry Department operates an inductively coupled plasma (ICP) machine that seemed wellsuited to analyzing the samples. However, it was discovered that this instrument was not able to detect certain atomic species in small quantities, such as rubidium. In addition, despite having a graduate student assigned full-time to its upkeep, the machine broke frequently. For these reasons, characterizing the cofactor samples-an essential step to this research-has been problematic and unreliable.

Conclusion

During this project, the author learned much concerning biochemistry, nitrogenases in particular, and how good scientific research is conducted. The protocol for isolating and purifying iron-molybdenum cofactor was refined and improved. Unfortunately, no reliable results, which could answer the original research question on cofactor structure and complexation properties, were obtained. The author plans more investigation toward this end. Future research may utilize an alternative method for characterizing the cofactor products, such as the proton induced x-ray emission (PIXE) machine operated by the BYU Physics Department and other instruments and techniques.

Research project completed as described above on September 2, 1994.