Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 5, Pages 1451-1455Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0610975104
Keywords
intermediate; kinetic isotope effect
Categories
Funding
- NHLBI NIH HHS [HL13531, R01 HL013531] Funding Source: Medline
- NIGMS NIH HHS [R01-GM59087, R01 GM059087] Funding Source: Medline
Ask authors/readers for more resources
A major obstacle to understanding the reduction of N-2 to NH3 by nitrogenase has been the impossibility of synchronizing electron delivery to the MoFe protein for generation of specific enzymatic intermediates. When an intermediate is trapped without synchronous electron delivery, the number of electrons, n, it has accumulated is unknown. Consequently, the intermediate is untethered from kinetic schemes for reduction, which are indexed by n. We show that a trapped intermediate itself provides a synchronously prepared initial state, and its relaxation to the resting state at 253 K, conditions that prevent electron delivery to MoFe protein, can be analyzed to reveal n and the nature of the relaxation reactions. The approach is applied to the H+/H- intermediate (A) that appears during turnover both in the presence and absence of N2 substrate. A exhibits an S = 1/2 EPR signal from the active-site iron-molybdenum cofactor (FeMo-co) to which are bound at least two hydrides/protons. A undergoes two-step relaxation to the resting state (C): A -> B -> C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE approximate to 3-4 (85% D2O). In the context of the Lowe-Thorneley kinetic scheme for N-2 reduction, these results provide powerful evidence that H-2 is formed in both relaxation steps, that A is the catalytically central state that is activated for N-2 binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available