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![Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase](/_next/image?url=https%3A%2F%2Fcloudfront.jove.com%2FCDNSource%2Fteasers%2F55858.jpg&w=3840&q=50)
10:01
Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase
Published on: December 4, 2017
A substrate channel in the nitrogenase MoFe protein
Brett M Barney1, Michael G Yurth, Patricia C Dos Santos
1Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322, USA.
Summary
Investigating the nitrogenase enzyme
Area of Science:
- Biochemistry
- Enzymology
- Nitrogen Fixation
Background:
- Nitrogenase facilitates the conversion of N2 to ammonia via the FeMo cofactor.
- The FeMo cofactor is deeply embedded within the MoFe protein, lacking direct substrate access.
- A water-filled channel connects the solvent to the FeMo cofactor's vicinity.
Purpose of the Study:
- To investigate the role of the water-filled channel in substrate and product transport.
- To assess the impact of channel amino acid substitutions on nitrogenase kinetics.
Main Methods:
- Site-directed mutagenesis of four amino acids lining the channel.
- Purification of engineered MoFe protein variants.
- Kinetic analysis of N2, acetylene, azide, and propyne reduction.
Main Results:
- Maximal reaction velocities (Vmax) remained largely unaffected by substitutions.
- Michaelis constants (Km) increased significantly (up to 22-fold) for tested substrates.
- Substitutions distant from the active site altered substrate binding affinity.
Conclusions:
- The water-filled channel is crucial for efficient substrate access to the FeMo cofactor.
- Channel modifications impact substrate binding rather than electron transfer rates.
- The channel functions as a dedicated pathway for substrates and products in nitrogenase.
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