Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From <em>Gluconacetobacter diazotrophicus</em>

Title

Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From Gluconacetobacter diazotrophicus

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Description

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensice crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding.

Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed herterlogously in non-diazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase.

ISBN

9780128147177

Publication Date

2018

Publisher

Academic Press

City

Cambridge, MA

Disciplines

Amino Acids, Peptides, and Proteins | Environmental Chemistry | Enzymes and Coenzymes | Laboratory and Basic Science Research | Organic Chemistry | Other Chemistry | Physical Chemistry | Research Methods in Life Sciences

Comments

In Sheila S. David (Ed.), Methods in Enzymology: Fe-S Cluster Enzymes Part B. Dr. Owens' chapter begins on page 355.

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Elsevier

Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From <em>Gluconacetobacter diazotrophicus</em>

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