B.S., 1983, University of Redlands, California Ph.D., 1989, University of California, Riverside Postdoctoral, 1989-93, University of Georgia

Biochemistry

Our laboratory is using multidisciplinary approaches including molecular biology, biochemistry, and biophysics to elucidate a molecular understanding of the mechanism of the metalloenzyme nitrogenase.

Nitrogenase is the enzyme responsible for all biological nitrogen fixation, a central reaction in the global nitrogen cycle. The reduction of N2 to NH3 catalyzed by nitrogenase can be summarized by the equation:

N2 + 8 H+ + 8 e- +16 MgATP —> 2 NH3 + H2 + 16 MgADP + 16 Pi

Biological nitrogen fixation accounts for approximately 80 % of the total input of fixed nitrogen into the biosphere each year (in excess of 2 x 1014 grams/year) and thus is essential to all living systems.

Given the significance of the reaction catalyzed by nitrogenase, there is considerable interest in understanding how the enzyme works. Nitrogenase is a complex metalloproteins that contains three different metal-based centers. A general diagram of the participation of the two nitrogenase proteins, ATP, electrons, and the metal centers is shown in the scheme.

We are working towards providing answers to four outstanding questions regarding how nitrogenase works: 1) How does N2 bind to the nitrogenase active site, 2) How are electrons delivered to the active site for N2 reduction, 3) What are the roles for MgATP hydrolysis in the reaction, and 4) How are the unusual metal centers of nitrogenase biosynthesized. To gain answers to these questions, we are employing a range of approaches including site-directed mutagenesis, EPR, rapid kinetics, NMR, CD, and x-ray spectroscopy. Students working in the group gain first hand training in these and other methods that are ideal for pursuing jobs in industry or academics.

Willis, R. M., McCurdy, A. T., Ogborn, M. K., Wahlen, B. D., Quinn, J. C., Pease III, L. F., and Seefeldt, L. C. (2014) Improving energetics of triacylglyceride extraction from wet oleaginous microbes. Bioresource Technology 167, 416–424. doi: 10.1016/j.biortech.2014.06.013

Shaw, S., Lukoyanov, D., Danyal, K., Dean, D. R., Hoffman, B. M., and Seefeldt, L. C. (2014) Nitrite and hydroxylamine as nitrogenase substrates: mechanistic implications for the pathway of N2 reduction. J. Am. Chem. Soc. 136, 12776–12783. doi: 10.1021/ja507123d

Willis, R. M., McCurdy, A. T., Ogborn, M. K., Wahlen, B. D., Quinn, J. C., Pease III, L. F., and Seefeldt, L. C. (2014) Improving energetics of triacylglyceride extraction from wet oleaginous microbes. Bioresource Technology 167, 416–424. doi: 10.1016/j.biortech.2014.06.013

Smith, D., Danyal, K., Raugei, S., and Seefeldt, L. C. (2014) Substrate channel in nitrogenase revealed by a molecular dynamics approach. Biochemistry 53, 2278–2285. doi: 10.1021/bi401313j

Lukoyanov, D., Yang, Z.-Y., Duval, S., Danyal, K., Dean, D. R., Seefeldt, L. C., and Hoffman, B. M. (2014) A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates. Inorg. Chem. 53, 3688–3693. doi: 10.1021/ic500013c

Hoffman, B. M., Lukoyanov, D., Yang, Z.-Y., Dean, D. R., and Seefeldt, L. C. (2014) Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062. doi: 10.1021/cr400641x

Yang, Z.-Y., Khadka, N., Lukoyanov, D., Hoffman, B. M., Dean, D. R., and Seefeldt, L. C. (2013) On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase. Proc. Natl. Acad. Sci. U.S.A 110, 16327–16332.

Wahlen, B. D., Morgan, M. R., McCurdy, A. T., Willis, R. M., Morgan, M. D., Dye, D. J., Bugbee, B., Wood, B. D., and Seefeldt, L. C. (2013) Biodiesel from microalgae, yeast, and bacteria: engine performance and exhaust emissions. Energy Fuels 27, 220–228.

Seefeldt, L. C., Yang, Z.-Y., Duval, S., and Dean, D. R. (2013) Nitrogenase reduction of carbon-containing compounds. Biochim. Biophys. Acta 1827, 1102–1111.

Moure, V. R., Danyal, K., Yang, Z.-Y., Wendroth, S., Müller-Santos, M., Pedrosa, F. O., Scarduelli, M., Gerhardt, E. C. M., Huergo, L. F., Souza, E. M., and Seefeldt, L. C. (2013) The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB. J. Bacteriol. 195, 279–286.

Hoffman, B. M., Lukoyanov, D., Dean, D. R., and Seefeldt, L. C. (2013) Nitrogenase: a draft mechanism. Acc. Chem. Res. 46, 587–595.

Duval, S., Danyal, K., Shaw, S., Lytle, A. K., Dean, D. R., Hoffman, B. M., Antony, E., and Seefeldt, L. C. (2013) Electron transfer precedes ATP hydrolysis during nitrogenase catalysis. Proc. Natl. Acad. Sci. U.S.A 110, 16414–16419.

Appel, A. M., Bercaw, J. E., Bocarsly, A. B., Dobbek, H., DuBois, D. L., Dupuis, M., Ferry, J. G., Fujita, E., Hille, R., Kenis, P. J. A., Kerfeld, C. A., Morris, R. H., Peden, C. H. F., Portis, A. R., Ragsdale, S. W., Rauchfuss, T. B., Reek, J. N. H., Seefeldt, L. C., Thauer, R. K., and Waldrop, G. L. (2013) Frontiers, opportunities, and challenges in biochemical and chemical catalysis of CO2 fixation. Chem. Rev. 113, 6621–6658.

Adams, C., Godfrey, V., Wahlen, B., Seefeldt, L., and Bugbee, B. (2013) Understanding precision nitrogen stress to optimize the growth and lipid content tradeoff in oleaginous green microalgae. Bioresour. Technol. 131, 188–194.

Yang, Z.-Y., Moure, V. R., Dean, D. R., and Seefeldt, L. C. (2012) Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase. Proc. Natl. Acad. Sci. U.S.A. 109, 19644–19648.

Seefeldt, L. C., Hoffman, B. M., and Dean, D. R. (2012) Electron transfer in nitrogenase catalysis. Curr. Opin. Chem. Biol. 16, 19–25.

Mayweather, D., Danyal, K., Dean, D. R., Seefeldt, L. C., and Hoffman, B. M. (2012) Temperature invariance of the nitrogenase electron transfer mechanism. Biochemistry 51, 8391–8398.

Lukoyanov, D., Yang, Z.-Y., Barney, B. M., Dean, D. R., Seefeldt, L. C., and Hoffman, B. M. (2012) Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase. Proc. Natl. Acad. Sci. U.S.A. 109, 5583–5587.

George, S. J., Barney, B. M., Mitra, D., Igarashi, R. Y., Guo, Y., Dean, D. R., Cramer, S. P., and Seefeldt, L. C. (2012) EXAFS and NRVS reveal a conformational distortion of the FeMo-cofactor in the MoFe nitrogenase propargyl alcohol complex. J. Inorg. Biochem. 112, 85–92.

Barney, B. M., Wahlen, B. D., Garner, E., Wei, J., and Seefeldt, L. C. (2012) Differences in substrate specificities of five bacterial wax ester synthases. Appl. Environ. Microbiol. 78, 5734–5745.