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Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Published on: December 19, 2011
An organometallic intermediate during alkyne reduction by nitrogenase
Hong-In Lee1, Robert Y Igarashi, Mikhail Laryukhin
1Department of Chemistry Education, Kyungpook National University, Daegu 702-701, Korea. leehi@knu.ac.kr
Journal of the American Chemical Society
|August 5, 2004
Summary
Researchers studied a nitrogenase enzyme intermediate using advanced electron-nuclear double resonance (ENDOR) techniques. This study reveals the structure of a trapped enzyme-reduction intermediate, offering insights into nitrogen fixation mechanisms.
Area of Science:
- Biochemistry
- Bioinorganic Chemistry
- Enzymology
Background:
- Nitrogenase is a crucial metalloenzyme catalyzing nitrogen (N2) reduction.
- Specific substitutions in nitrogenase MoFe protein alter substrate range.
- Understanding nitrogenase intermediates is key to nitrogen fixation.
Purpose of the Study:
- To characterize a trapped S = 1/2 intermediate of nitrogenase MoFe protein during propargyl alcohol reduction.
- To elucidate the structure of the active-site FeMo-cofactor intermediate using isotopic labeling and advanced ENDOR techniques.
Main Methods:
- Site-directed mutagenesis of nitrogenase MoFe protein (alpha-70 Val to Ala).
- Preparation of isotopically substituted propargyl alcohol ((13)C, (1,2)H).
- Application of continuous wave (CW) and pulsed (13)C ENDOR, Mims pulsed ENDOR, and stochastic field-modulated ENDOR at 35 GHz.
Main Results:
- A trapped S = 1/2 turnover intermediate was generated and studied.
- The intermediate contains the 3-carbon chain of propargyl alcohol with resolved (1,2)H ENDOR signals from three protons.
- Two strongly coupled protons (H(a)) exhibit identical hyperfine tensors, constraining structural models.
- A novel bio-organometallic complex is proposed, with a reduced propargyl alcohol product bound as a metalla-cyclopropane to an Fe atom.
Conclusions:
- The study provides the first detailed description of a trapped nitrogenase reduction intermediate.
- The proposed structure, a metalla-cyclopropane complex, offers a plausible model for substrate binding and reduction.
- This work advances the understanding of the intricate mechanisms of nitrogen fixation.
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