Nitrogenase reactivity: cyanide as substrate and inhibitor

Area of Science:

• Biochemistry
• Enzymology
• Nitrogen Fixation Research

Background:

• Nitrogenase enzymes catalyze essential biological reductions.
• Understanding nitrogenase component proteins (Av1 and Av2) interactions is crucial.
• Previous studies suggested self-inhibition in cyanide reduction by nitrogenase.

Purpose of the Study:

• To investigate the mechanism of cyanide reduction by purified nitrogenase component proteins (Av1 and Av2).
• To clarify the roles of different cyanide species (CN- and HCN) in the reaction.
• To identify the substrate and products of cyanide reduction and elucidate the inhibition mechanism.

Main Methods:

• Enzymatic assays using purified Av1 and Av2 proteins.
• Kinetic analysis to determine inhibition constants (Ki) and Michaelis constants (Km).
• Product analysis and electron balance studies.
• Investigation of effects of various substrates and inhibitors (CO, azide, N2, C2H2, N2O, H2).

Main Results:

• The previously reported self-inhibition was identified as an artifact.
• Cyanide ion (CN-) acts as a potent reversible inhibitor, uncoupling MgATP hydrolysis and electron transfer (Ki = 27 microM).
• Hydrogen cyanide (HCN) is the actual substrate for reduction (Km = 4.5 mM).
• Reduction products include methane and ammonia (6e-), and methylamine (4e-).
• HCN reduction occurs at a more oxidized enzyme state than N2 reduction or H2 evolution.
• Nitrous oxide and acetylene influence product distribution, suggesting simultaneous binding.

Conclusions:

• CN- is a reversible inhibitor of nitrogenase-mediated electron flow, not a substrate.
• HCN is the substrate for cyanide reduction, producing methane and ammonia.
• The findings provide new insights into the complex reaction mechanisms of nitrogenase.