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

Abstract

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N) to ammonia (NH). 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 extensive 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 heterologously in nondiazotrophic 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.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

“…We chose to study CowN in the diazotroph Gluconacetobacter diazotrophicus , an agriculturally relevant organism with a well-characterized nitrogenase ( 30 , 31 , 32 ). Importantly, G. diazotrophicus has no alternative nitrogenases so any regulatory cross talk between nitrogenases that may influence CowN expression can be ruled out.…”

“…Nitrogenase was expressed and purified based on previously described methods ( Fig. S7 ) ( 30 ). Our typical maximum MoFeP C 2 H 2 reduction activity is about 1500 nmol mg −1 min −1 ( Fig.…”

“…G. diazotrophicus was grown in LGI media ( 30 ) containing 5 mM potassium phosphate, pH 6, and 0.5 mM (NH 4 ) 2 SO 4 . Cell were grown in 6 L Erlenmeyer flasks that were filled to 2.5 L. Growth was started by adding 20 mL of a starter culture (OD 600nm ∼ 1) to each flask, and cultures were grown at 30 °C with a shaker speed of 200 rpm.…”

“…Cell were grown in 6 L Erlenmeyer flasks that were filled to 2.5 L. Growth was started by adding 20 mL of a starter culture (OD 600nm 1) to each flask, and cultures were grown at 30 C with a shaker speed of 200 rpm. After about 4 to 5 days, the optical density reached 0.6 to 0.8, at which point nitrogenase in G. diazotrophicus became derepressed and nitrogenase activity could be observed using acetylene reduction assays, as described previously (30). Cells were harvested at an OD 600nm 1.0 to 1.4 by centrifugation at 5000 rpm and stored at −80 C until use.…”

Artificial, Photoinduced Activation of Nitrogenase Using Directed and Mediated Electron Transfer Processes

“…It has been shown that the Fe protein binds to nitrogenase via a positively charged docking site. [40][41][42] We hypothesized that TGA-CdS NPs with negative charges on their surface will bind to the docking site and force improved electron transfer process.…”