Nitrogenase Caught in Action

Enzymology

In nature, enzymes known as nitrogenases convert N₂ found in the environment into a form that can be used to make amino acids and other biologically valuable nitrogen compounds. A multidisciplinary team has now trapped N₂-derived intermediates bound to a nitrogenase. This long-awaited achievement, the researchers say, may open the door to understanding how these enzymes work.

The structure of nitrogenase and its active site has been known for more than a decade, notes spectroscopist Brian M. Hoffman of Northwestern University. A complex metal cofactor in the multisubunit enzyme reduces N₂ to NH₃. But this structural information told us nothing about how this enzyme is able to bind and reduce substrates, Hoffman points out.

For years, Hoffman has been working with biologist Dennis R. Dean of Virginia Tech and biochemist Lance C. Seefeldt of Utah State University to figure out how to trap and characterize nitrogenase intermediates that might shed light on the enzyme's mechanism. Now they've succeeded in trapping the enzyme with N₂-derived intermediates bound to the active site (J. Am. Chem. Soc. 2005, 127, 14960). This is the break needed to get at the mechanism of this complex metalloenzyme, Seefeldt says.

Guided by their previous experiments with nonphysiological substrates, the researchers exposed subtly modified versions of the enzyme to N₂ or one of two suspected dinitrogen intermediates and flash-froze the samples. Spectroscopy confirmed that they had indeed captured enzyme-bound intermediates.

The team is now carrying out more spectroscopic work to deduce the structure of the trapped enzyme-bound intermediates. That work should shed light on the most pressing questions about how nitrogenase works, including where N₂ binds to the active site and how it is reduced, Hoffman says.