Decoding the nitrogenase mechanism: the homologue approach

Area of Science:

• Biochemistry
• Enzymology
• Nitrogen Cycle

Background:

• Nitrogenase (Mo)-nitrogenase catalyzes crucial dinitrogen (N(2)) to ammonia (NH(3)) conversion.
• Understanding nitrogenase's catalytic mechanism is hindered by its redox-active iron-molybdenum cofactor (FeMoco).
• FeMoco's rapid, unsynchronized redox cycling complicates capturing catalytic intermediates.

Purpose of the Study:

• To overcome challenges in studying nitrogenase mechanism by using a slower, partially defective homologue.
• To investigate substrate-enzyme interactions and catalytic steps in nitrogen fixation.
• To establish a platform for reconstructing a functional nitrogenase MoFe protein.

Main Methods:

• Utilized the NifEN protein complex, a nitrogenase homologue with slower turnover.
• Analyzed electron paramagnetic resonance (EPR) signals during acetylene (C(2)H(2)) turnover.
• Employed a mutational strategy to gradually introduce MoFe protein features into NifEN.

Main Results:

• Observed a new S = 1/2 EPR signal in NifEN during C(2)H(2) turnover, suggesting a stabilized intermediate.
• The defective NifEN homologue facilitates the potential crystallization of an intermediate-bound nitrogenase.
• NifEN's unique properties enable step-by-step functional analysis of MoFe protein components.

Conclusions:

• The NifEN homologue provides a novel approach to study nitrogenase mechanism and capture catalytic intermediates.
• This strategy aids in defining the roles of specific components like the P-cluster and cofactor in nitrogen fixation.
• Further research using NifEN can elucidate the complete mechanism of biological nitrogen fixation.