Trapping an intermediate of dinitrogen (N2) reduction on nitrogenase

Area of Science:

• Biochemistry
• Enzymology
• Bioinorganic Chemistry

Background:

• Nitrogenase catalyzes the reduction of dinitrogen (N2) to ammonia, a crucial step in the biological nitrogen cycle.
• Understanding the mechanism of N2 reduction by nitrogenase is vital for agricultural and industrial applications.
• Previous studies have characterized intermediates using modified MoFe proteins, but trapping intermediates with wild-type enzymes remains challenging.

Purpose of the Study:

• To identify and characterize an intermediate formed during the reduction of N2 by wild-type nitrogenase.
• To provide direct evidence for the binding of N2 to the active-site FeMo cofactor during catalysis.
• To elucidate the mechanism of N2 substrate binding and reduction by nitrogenase.

Main Methods:

• Steady-state turnover of wild-type MoFe protein with N2 at 77 K.
• Electron Paramagnetic Resonance (EPR) spectroscopy to detect and characterize intermediates.
• 15N-ENDOR (Electron-nuclear double resonance) spectroscopy to confirm the presence and binding of 15N2.
• Varying N2 partial pressure, Fe protein to MoFe protein ratio, and pH to optimize signal detection.

Main Results:

• A novel EPR signal (S=1/2, g=[2.08, 1.99, 1.97]) was observed, dependent on N2 partial pressure and electron flux.
• This N2-dependent signal replaced the resting-state FeMo cofactor EPR signal (S=3/2), indicating enzyme reduction.
• 15N-ENDOR spectroscopy confirmed the presence of a bound 15N2 molecule to the FeMo cofactor, likely end-on, with specific hyperfine couplings.

Conclusions:

• A high-concentration intermediate containing N2 bound to the FeMo cofactor was trapped during wild-type nitrogenase turnover.
• This intermediate is distinct from those observed with non-natural substrates or modified enzymes.
• The findings provide direct spectroscopic evidence for end-on N2 binding to the FeMo cofactor, advancing mechanistic insights into nitrogen fixation.