“Towards the Mechanism of Nitrogenase”

By: Steven Reinhardt

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee

3210 North Cramer Street, Milwaukee, WI 53211

Advisor: Dr. Arsenio Pacheco

ABSTRACT

Reduced nitrogen is an essential component for all organisms, required in both amino and nucleic acid structures. Despite the atmosphere consisting of 79% N2, it is essentially inert, and unavailable to biosynthetic processes. Geological sources of reduced nitrogen such as lightning and volcanos only provide a small fraction of the world total, the primary sources of reduced nitrogen are fixation by a small set of bacteria, and the industrial Haber-Bosch process for production of ammonia. Biological fixation of nitrogen, the reduction of N2 to 2 NH3 is performed by the enzyme nitrogenase. This enzyme consists of two proteins, an Fe electron transfer protein, and the catalytic active MoFe protein. Studies over the years have given us a large amount of information about the structures of the two proteins, alternate substrates reduced by nitrogenase, and a solid kinetic foundation including rate constants for the intermediate states of catalysis in the form of the Lowe-Thorneley model.[1] Unfortunately, a mechanism has been lacking because identification of the intermediates had remained elusive. Recently though, the use of experimental techniques of freeze-quenching and quench-cryoannealing were used to trap catalytic intermediates, which were then combined with EPR and related techniques, to enable the characterization of several of these species. Of significant importance is the determination of the E4 intermediate, named the Janus intermediate, whose structure leads to an explain of how the active site can reduce dinitrogen.[2] Analysis of the intermediates within the framework of several proposed mechanisms based on inorganic complexes has allowed the formation of a complete mechanism for nitrogenase within the context of established experimental and kinetic results.[3]

[1] Burgess, B. K.; Lowe, D. J. Chem. Rev. 1996, 96, 2983.

[2] Hoffman, B. M.; Lukoyanov, D.; Dean, D. R.; Seefeldt, L. C. Acc. Chem. Res. 2013, 46, 587.

[3] Hoffman, B. M.; Lukoyanov, D.; Yang, Z. Y.; Dean, D. R.; Seefeldt, L. C. Chem. Rev. 2014, 114, 4041.