Connecting nitrogenase intermediates with the kinetic scheme for N₂ reduction by a relaxation protocol and identification of the N₂ binding state
2007
Lukoyanov, Dmitriy | Barney, Brett M. | Dean, Dennis R. | Seefeldt, Lance C. | Hoffman, Brian M.
A major obstacle to understanding the reduction of N₂ 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 = [fraction one-half] 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 [rightward arrow] B [rightward arrow] C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE [almost equal to] 3-4 (85% D₂O). In the context of the Lowe-Thorneley kinetic scheme for N₂ reduction, these results provide powerful evidence that H₂ is formed in both relaxation steps, that A is the catalytically central state that is activated for N₂ binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons.
Show more [+] Less [-]AGROVOC Keywords
Bibliographic information
This bibliographic record has been provided by National Agricultural Library
